Age determination of body fluids by mass spectrometry - … · Age determination of body fluids by...

Age determination of body fluids by

mass spectrometry

Katarzyna Szykuła

Master Thesis

2015 – 2016

2

MSc Chemistry

Analytical Sciences

Master Thesis

Age determination of body fluids by mass spectrometry

by

Katarzyna Szykuła

June 2016

Supervisor: Second examiner:

Prof. dr. Garry Corthals Dr. Michelle Camenzuli

Analytical Chemistry Group

van’ t Hoff Institute for Molecular Sciences

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Acknowledgements

I would like to express my gratitude to professor Garry Corthals for his supervision and guidance

during this Master’s project. I have enjoyed my time carrying out this research and I would like to

thank everyone from the Analytical Chemistry group for their help and kindness. Additionally, I would

like to thank Dr. Michelle Camenzuli for reviewing this thesis.

Moreover, I would like to thank professor Maurice Aalders and his group for their help and the

opportunity to do experiments at Academic Medical Center.

4

Abstract

In forensic science, biological traces have a significant importance for forensic investigators. They can

provide crucial information about the donor or about a crime itself, for example what happened on a

crime scene or when the criminal incident was committed. In order to estimate the latter one, aging

of body fluids found on a crime scene is studied. This research investigated aging process of four

body fluids: blood, saliva, semen and fingermarks. As all of them are protein-containing fluids, this

type of compounds was examined by using mass spectrometry. Samples of aforementioned body

fluids were analyzed at different age, and subsequently, the numbers of proteins identified as well as

their quantification were compared. For blood, saliva and semen, the number of unique proteins was

changing with the age of a sample. Additionally, some proteins showed variations in quantity when

sample was getting older. These findings can indicate that protein levels are changing over time in

biological traces, and these biomolecules can be potential biomarkers for age estimation of specific

body fluid found on a crime scene.

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Table of Contents

Acknowledgements ................................................................................................................................. 3

Abstract ................................................................................................................................................... 4

1. Introduction ......................................................................................................................................... 7

1.1 Forensic biological traces .............................................................................................................. 7

1.1.1 Blood ....................................................................................................................................... 7

1.1.2 Saliva ....................................................................................................................................... 8

1.1.3 Semen ..................................................................................................................................... 8

1.1.4 Fingermarks ............................................................................................................................ 8

1.2 Mass spectrometry ........................................................................................................................ 9

1.2.1 Proteomics .............................................................................................................................. 9

1.2.2 Quantitation in proteomics .................................................................................................. 11

1.2.3 Proteomics in forensic science ............................................................................................. 13

2. Materials and methods ..................................................................................................................... 15

2.1 Materials ...................................................................................................................................... 15

2.2 Methods ...................................................................................................................................... 15

2.2.1 Blood ..................................................................................................................................... 15

2.2.2 Saliva ..................................................................................................................................... 17

2.2.3 Semen ................................................................................................................................... 17

2.2.4 Fingermarks .......................................................................................................................... 17

3. Results and Discussion ...................................................................................................................... 19

3.1 Blood............................................................................................................................................ 19

3.1.1 Method optimization ............................................................................................................ 19

3.1.2 Examining aging of blood ..................................................................................................... 20

3.2 Saliva ............................................................................................................................................ 25

3.3 Semen .......................................................................................................................................... 30

3.4 Fingermarks ................................................................................................................................. 34

4. Conclusions and future research ....................................................................................................... 35

5. References ......................................................................................................................................... 36

Appendix 1. ............................................................................................................................................ 39

Appendix 2. ............................................................................................................................................ 44

Appendix 3. ............................................................................................................................................ 51

Appendix 4. ............................................................................................................................................ 57

Appendix 5. ............................................................................................................................................ 61

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Appendix 6. ............................................................................................................................................ 65

Appendix 7. ............................................................................................................................................ 70

Appendix 8. ............................................................................................................................................ 76

Appendix 9. ............................................................................................................................................ 82

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1. Introduction

orensic science is an enormously developing field nowadays. As an interdisciplinary study, it

utilizes the knowledge and technology from several fields, such as chemistry, physics, medicine

and mathematics to solve crimes. On a crime scene, investigators can find many forensically

important traces. Traces like: pieces of glass, hairs, fibers or paints are defined as physical traces. On

the other hand, body fluids, which can be commonly found on a crime scene as well, belong to the

group of biological traces. All traces found on a crime scene can play a crucial role during

investigations. They can provide information about the suspect/victim or what happened during the

criminal incident. However, this thesis will focus only on body fluids, therefore, only biological

evidence will be further described in details.

Examining biological evidence has a high importance for investigators as they can provide

intelligence about the donor (by DNA profiling) or about the crime of investigation. The latter one is

often studied by analyzing the bloodstain pattern, which can give the information about events that

occurred on the crime scene [1]. Moreover, by studying the aging of body fluids, it can be estimated

when a crime took place, which can narrow down the time intervals for detectives. To date, several

attempts have been performed to study body fluids aging by using mainly spectroscopic [2, 3] or

chromatographic methods [4, 5]. However, these studies did not provide with sufficient information

about the aging process. Therefore, the aim of this research was to use mass spectrometry

technique, suitable for trace analysis, to study the aging of body fluids.

1.1 Forensic biological traces

1.1.1 Blood

This body fluid is composed of blood cells (red cells, white cells and platelets) suspended in blood

plasma. The red color of blood is due to an iron-containing protein – hemoglobin, which its main

function is to transport the oxygen from lungs to tissues[6]. As blood contains proteins, lipids,

electrolytes, metabolites, hormones and more [6], this fluid is the most extensively studied, not only

in medical fields. In forensics, studying blood traces has various purposes. Besides obtaining DNA

profile, or studying blood stain pattern [1], also aging of this body fluid has been examined.

The idea to study blood aging is not new. Many attempts were made to investigate aging process by

studying different blood components with various methods, for example RNA with reverse

transcription polymerase chain reaction (RT-PCR) [7], erythrocytes with electron paramagnetic

resonance (EPR) [3] or globulins with immunoelectrophoresis [8]. More recent, three hemoglobin

derivatives: oxy-hemoglobin (oxy-Hb), methemoglobin (met-Hb) and hemichrome (HC) were studied

[2, 9, 10]. As the fractions of these derivatives are changing in time (fraction of oxy-Hb decreases and

met-Hb and HC increase), Bremmer et al.[2] applied reflectance spectroscopy to record spectra of

blood stains and calculated these fractions. They presented the potential of this method, as they

were able to discriminate age of blood stains with larger time intervals (e.g. 20 days). Edelman et al.

applied hyperspectral imaging to record the spectra of the described hemoglobin derivatives and

F

8

observed a decreased error compared to reflectance spectroscopy [10]. However, Bremmer et al. [9]

showed that conversion of oxy-Hb to met-Hb and HC is dependent on temperature and humidity.

Moreover, both methods were successful only when spectra were recorded against a white

background.

1.1.2 Saliva

This very essential body fluid has many functions, but the most important are: food solubilization,

lubrication, antibacterial, buffering and food digestion. Saliva is produced by salivary glands and it

contains water, electrolytes, proteins and other organic molecules [11]. Its chemical composition has

been studied widely, mostly for cancer [12], autoimmune diseases [13], hereditary diseases [14], or

viral diseases [15]. Saliva can also be used for drug analysis [16].

On a crime scene, this oral body fluid can be found for instance on food or cigarettes. In forensic

science, it is mainly studied for DNA profiling and aging studies have never been reported before.

Nevertheless, some study has shown that when saliva is in ex situ conditions [17], proteins can

degrade over time. This information is of importance and can indicate that saliva proteins have

potential to be used as a biomarker for age determination of saliva.

1.1.3 Semen

The main composition of semen is seminal fluid which may contain spermatozoa [18]. This fluid

contains high concentration of sugars (fructose and glucose) and proteins [19]. As study showed, in a

single individual seminal fluid, over 900 proteins can be identified [20] which can suggest that human

seminal fluid proteome can be used for biomarker studies, for example in cancer [21] or infertility

research[22]. In forensic science, this biological evidence can be found on a crime scene in a case of

sexual assaults. Semen has a high evidential importance as from this fluid donor DNA profile can be

obtained. For identification purposes, forensic investigators utilize the proteins’ natural fluorescence

from semen. However, there is yet no literature published to use this biomolecules in age

determination of seminal fluid.

1.1.4 Fingermarks

This type of traces is often found a crime scene. The chemical composition of fingermarks is diverse.

They contain elements (e.g. sodium, potassium, chloride), amino acids, proteins, fatty acids and

other lipids [23]. Forensic scientists study fingermarks to obtain as much information as possible

about the donors. For instance, from analysis of fingermarks information about the sex [24] or drug

abused [25] can be obtained. Similar to the other body fluids, the knowledge of the age of

fingermarks can be of significant importance for investigators. Wolstenholme et al. [26] attempted to

determine the age of fingermarks by investigating the degradation of olenaic acid. The other

research group, van Dam et al. [27], showed that the fluorescence signal is changing with the age of

the sample. However, the estimation error of aged samples was relatively high - only 55% of aged

male fingermarks estimated within the error of 1.9 days.

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1.2 Mass spectrometry

Mass spectrometry (MS) is a widely used method in analytical chemistry. Its very intense

development can be proved by five Nobel Prizes, starting from 1906 with J.J.Thomson for his work on

the electrical conductivity of gases. Subsequently, other prizes were for isotope discovery by mass

spectrograph, for cyclotron and for the development of the ion trap technique. The last Nobel Prize

in mass spectrometry, until now, was awarded to J. Bennet and K. Tanaka in 2002. The first one for

developing soft desorption ionization methods, and the second one for developing a new method for

mass spectrometric analyses of biological macromolecules; both extremely important in protein

analysis. Mass spectrometry, as a versatile technique, has found its application in various disciplines,

such as toxicology, forensic science, clinical or environmental chemistry. Its main advantage is high

sensitivity [28], which can be critical, especially, in forensic science for analyzing biological traces,

which often are found on the crime scene in scarce or mixed.

1.2.1 Proteomics

Proteomics is the field of science which studies the biological processes in organisms by protein

analysis. A proteome is the entire protein complement expressed by a genome. However, in contrast

to genes, the nature of proteins is not static but dynamic. Proteins can undergo modifications

(phosphorylation, oxidation), their abundance can change or they can form clusters. Those variations

are dependent on the physiological state of the organism. Therefore, the proteome reflects the state

of a cell or a tissue, and it is crucial to not only identify all the proteins but also to measure their

changes as well as pathways of change[29].

Protein analysis is a challenging task, as proteome is not only very complex because of all interactions

and modifications described before, but also, compared to genes, proteins cannot undergo

amplifications, which is the limiting factor when a sample amount is scarce. Hence, only appropriate

tools (i.e. high sensitivity) can be applied in proteomics, which makes mass spectrometry a suitable

method.

In proteomics, there are two ionization methods in widespread use: matrix-assisted laser

desorption/ionization (MALDI) and electrospray ionization (ESI). Both of them can generate ions from

large nonvolatile biomolecules. MALDI and ESI are knows as soft ionization methods, which means

that there is a minimal or no fragmentation of the analytes. The first one is mostly used for peptide

mass fingerprinting or tissue imaging. In MALDI, sample is mixed with a matrix, which molecules have

a strong absorption at the applied laser wavelength. The absorbed energy is transferred to the

analyte, which is desorbed and ionized together with matrix molecules. This solid-phase technique

mostly generates singly charged ions. Electrospray ionization is a liquid technique, which is easy to

couple to such separation methods as liquid chromatography or electrophoresis. Here, a high voltage

is applied, which generates highly charged droplets of the analyte. In contrast to MALDI, ESI

generates multiply charged ions [28].

The typical workflow in proteomics for LC-MS/MS analysis is presented on the Fig. 1. Briefly, proteins

in a sample undergo digestion into peptides, mostly by using enzyme digestion, trypsin for example.

Those peptides prior to MS/MS analysis are separated with reverse-phase liquid chromatography. In

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MS/MS analysis, the precursor ions as well as product ions are scanned, which provides crucial

information about the structure of a sample. After the analysis, by using appropriate software,

protein and peptide identification can be obtained.

Figure 1. Typical workflow in proteomics for LC-MS/MS analysis. Adapted from reference [30].

The instrument used in this project was TripleTOF mass spectrometer. The scheme below presents

the features in this instrument. Briefly, QJet and Q0 are used for ion focusing, Q1 selects the

precursor ion and Q2 is the collision cell, where the ions collide with neutral gas molecules

(nitrogen). That formed product ions leave the collision cell and are re-accelerated and focused by

ions optics into a parallel beam which constantly enters TOF analyzer. There, a pulsed electric field is

pushing ions orthogonally to their previous direction, and then they acquire their final energy [31].

The accelerated ions travel through the tube, where at the opposite site is an electrostatic ion mirror

which reverses the ions direction to the detector. Ions gain the same kinetic energy, however, their

velocities are different, due to the mass differences. And consequently, ions reach the detector at

different time.

MS1

MS2

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Figure 2. The scheme of triple TOF MS features. (a) The major platform features in details. (b) an image of this platform[32].

1.2.2 Quantitation in proteomics

Nowadays, only protein identification is no longer sufficient in proteomics studies. In order to gain

more knowledge about living organisms, tissues or cells, it is important to quantify the proteins. Their

abundances can change over a time or under specific conditions, such as disease for example. In

proteomics, there are two quantitative approaches, one which is based on labeling the sample and

the second one is label-free. Labeling techniques can be divided into three categories such as:

metabolic labeling, chemical labeling and addition of standard peptides. In those approaches, labeled

peptides have the same chemical properties as unlabeled ones, with the only difference in their

masses. In metabolic labeling, such as stable isotope labeling by amino acids in cell culture (SILAC) for

example, samples of “light” and “heavy” amino acids are mixed at early stage. In chemical labeling,

the label can be introduced at protein or peptide levels. One of commonly used chemical labeling is

the isotope-coded affinity tag (ICAT). This label is bound to sulfhydryl groups of cysteine residues and

is useful to study oxidation or reduction level of proteins. However, as cysteine belongs to low

abundant amino acids in proteins, it reduces the number of peptides that can be examined. The

other type of chemical labeling is isobaric mass tags. There are two tags commercially available:

tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ)[33]. The

third technique of labeling approach in quantitative proteomics is by using synthetic isotope-labeled

peptides as a standard for absolute quantification (AQUA). These reference peptides allow to precise

measure the level of proteins and posttranslationally modified proteins. For AQUA, selected reaction

monitoring (SRM) analysis is performed [34].

In label-free approach, two quantitation techniques can be distinguished. First one is based on under

the curve (AUC) or signal intensity measurements, both based on precursor ion spectra. With these

methods chromatographic alignment is required. The second technique, spectral counting, is based

on counting the number of peptides assigned to a protein in MS/MS analysis. In spectral counting

methods, more abundant peptides are selected for fragmentation, and they produce a higher

abundance of MS/MS spectra. This process is proportional to protein amount in data-dependent

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acquisition. In order to assess protein abundance in the analyzed sample, protein abundance index

(PAI) is used. This index is defined as the number of identified peptides divided by the number of

observable generated peptides for every protein within a preferred mass range [35]. In order to

obtain absolute quantitation, PAI can be converted into exponentially modified emPAI, which is

equally to exponential form of PAI minus one, and this is proportional to protein content in analyzed

samples [36]. As in spectral counting, longer proteins generate more peptides than smaller ones, this

also can be used for quantification. In this case, normalized spectral abundance factor (NSAF) is used

which takes into account the length of the protein. In order to calculate NSAF, the number of spectral

counts for protein is divided by its length and this is defined as spectral abundance factor (SAF),

which has to be normalized by division by the sum of all SAFs for all proteins in a mixture [37].

Before applying a specific quantitation method, it is essential to consider which parameters are

crucial in the performed analysis. For example, it has to be determined what proteome coverage is

desired in the analyzed sample. This and other parameters for quantitative methods are overviewed

in the table below.

Table 1. Overview of quantitation methods applied in proteomics [33].

Method Proteome coverage Quantitative accuracy Quantitation type

Metabolic labeling

SILAC Medium Precise Relative

Chemical labeling

ICAT Poor Precise Relative

iTRAQ, TMT Medium Medium Relative

Standard peptide Poor Precise Relative/Absolute

Label-free

Ion intensities (PCP) Good Medium Relative

Spectrum count Good Poor Relative

Derived indices (APEX, emPAI)

Good Poor Relative/Absolute

Figure 3 shows that from all proteins in a sample, only a certain amount can be identified.

Furthermore, from this amount, even a smaller fraction can be successfully quantified. It emphasizes

how challenging protein analysis is and how important it is to perform appropriate sample

preparation with suitable enrichment and purification steps. Additionally, it also indicates how much

of the proteome is still unknown.

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Figure 3. Graphical representation of the amount of proteins that can be identified relative to all proteins in a sample [33].

1.2.3 Proteomics in forensic science

In forensic science it is very important to identify as many traces as possible which can be found on a

crime scene. The information of the trace’s origin can facilitate to reconstruct what has happened on

a crime scene. Especially, very crucial is the analysis of biological matrices as they can contain DNA,

which can have a huge value for investigators. However, as mostly those matrices are the mixtures of

several body fluids, such analysis is a complicated process. To identify such mixtures, an advanced

and sensitive method, like mass spectrometry, is needed. With this method, such body fluids as

blood, saliva, semen, vaginal fluids, urine and nasal secretions can be identified by a protein-specific

biomarker, which is unique for every body fluid [38, 39]. Moreover, analyzing biological traces with

MS is not destructive for DNA, which is an another advantage [40]. Hence, identification of the

source of a biological trace can be performed prior to DNA analysis.

The other approach of applying proteomics in forensic field is investigating organ-specific proteome.

This studies are especially useful in a case of ballistic reconstruction of the event [41] as the

information about which organ was damaged with the bullet can help to reconstruct the bullet’s

projectile.

Table 2. Organ-specific proteins [41].

Organ Protein

Heart

myosin binding protein C,cardiac-type glycogen phosphorylase, brain form troponin I, cardiac muscle myosin light chain 3 pyruvate dehydrogenase E1 component subunit beta, mitochondrial calpastatin NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4 cytochrome c oxidase subunit 4 isoform 1, mitochondrial

Kidney

calbindin Na(+)/H(+) exchange regulatory cofactor NHE-RF3 low-density lipoprotein receptor-related protein 2 villin 1 retinyl ester hydrolase type 1 membrane metalloendopeptidase variant 2 phosphotriesteraserelated protein plastin-1

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Liver

hydroxymethylglutaryl- CoA synthase, mitochondrial carbamoyl-phosphate synthase [ammonia], mitochondrial phenylalanine-4- hydroxylase 3-oxo-5-beta-steroid 4-dehydrogenase catechol Omethyltransferase acetyl-CoA acetyltransferase cytochrome P450 2E1 dimethylaniline monooxygenase

Lung

plastin-2 cathelicidin-4 tubulin beta chain cysteine and glycine-rich protein 1 prostaglandin F synthase 2 myosin light polypeptide 6 calpain-2 catalytic subunit alpha-actinin-1

Muscle

bridging integrator 1 myosin-binding protein C, slow-type fructose-1,6- bisphosphatase isozyme 2 myosin-binding protein C, fast-type troponin C, skeletal muscle (fast type) myosin regulatory light chain 2, skeletal muscle isoform nebulin PDZ and LIM domain protein 3

As it can be seen, protein studies by mass spectrometry are common used in forensic science. It can

provide with very substantial information about the donor of a sample, or about the circumstances

that the crime happened. However, one of the very important information obtained from body fluids

has not been yet investigated using mass spectrometry – age determination of body fluids by protein

analysis. This thesis presents a novel approach of studying the aging process with LC-MS/MS method.

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2. Materials and methods

2.1 Materials

All samples were taken from healthy donors and kept in the dark at room temperature.

Chemicals:

Acetonitrile, methanol, water, formic acid (Biosolve)

Ammonium bicarbonate, DTT, iodoacetamide, Tris(2-carboxyethyl)phosphine hydrochloride, sodium

deoxycholate, Brilliant Blue G (Sigma Aldrich)

Sequencing Grade Modified Trypsin (Promega)

4-20% Tris-glycine precast gel, Tricine-Glycine SDS Running Buffer (Novex)

4-15% Tris-HCl precast gel (Biorad)

Apparatus:

Samples were collected on: Human ID Bloodstain Card; glass microfibers filter (Whatman) or into 2.0

ml Eppendorf vials.

Solid-phase extraction was performed with C18 solid-phase extraction cartridges, 4mm/1mL

(Empore).

LC system consist of nanoLC 425; separation column C18, 3µm, 120Å, 0.075x150mm, and trap

column C18, 3µm, 120Å, 350µmx0.5mm (Eksigent). Flow rate: 300nL/minute, injection volume 1-5

µL.

MS/MS analysis was performed with Triple TOF 5600+ (ABSciex) in positive mode. TOF m/z range:

400-1250, product ion m/z range 100-2000.

2.2 Methods

2.2.1 Blood

Dried blood spots were prepared by spotting 15µl of blood on Whatman filter paper. Samples

preparation for LC-MS/MS analysis was performed according to the protocol described by Chambers,

A.G., et al. [42]. Briefly, dried blood spots were excised from the filter paper and transferred to 1.5 ml

Eppendorf tubes. Fresh sample (15µl) was pipetted directly to the vial. In order to dissolve dried

blood spots and dilute fresh blood, 800µl of 25mM ammonium bicarbonate was added to the vials

and vortexed. Subsequently, 100 µl of 10% sodium deoxycholate and 10 µl of 0.5 M Tris(2-

carboxyethyl)phosphine hydrochloride were added and samples were incubated for an hour at 60°C.

Then, samples were alkylated for one hour in the dark (covered with aluminum foil) in room

temperature after addition of 52.63µl 200mM iodoacetamide. In order to consume remain

iodoacetamide, 55.4µl of 200mM dithiotreitol (DTT) was added and samples were incubated for 30

minutes at 37°C. Next, 10 µl of trypsin solution was added and protein digestion was performed

16

overnight at 37°C. Protein concentration in samples, before digestion, was estimated with Bradford

assay (Biorad). After digestion, sodium deoxycholate was precipitated by adding 40 µl of 2% formic

acid and the samples were centrifuged at 4000 g for 20 minutes. The supernatant was collected into

the new vials. Prior to LC-MS/MS analysis, samples were desalted by using solid-phase extraction.

Cartridges were first pre-washed twice with 50% ACN/water and 0.1% TFA. Afterwards, samples

were loaded and washed with 0.1% TFA. Analytes were eluted with 0.1% formic acid in 60% ACN.

The collected samples were, subsequently, evaporated to dryness and peptides were reconstituted

with water to obtain a final protein concentration of 0.2µg/µl.

In order to obtain the highest number of protein identification, three different LC methods were first

tested with fresh blood samples. For all methods, the same chemicals were used; mobile phase A:

0.1% formic acid, mobile phase B: 100% acetonitrile.

Table 3. Gradient profile for method 1 [43].

Time [min] Mobile phase A% Mobile phase B%

0 97.3 2.7

1.5 93.7 6.3

16 86.5 13.5

18 86.2 13.8

33 77.5 22.5

38 59.5 40.5

39 19 81

42.9 19 81

43 97.3 2.7

Table 4. Gradient profile for method 2.


0 100 0

10 95 5

40 40 60

45 20 80

60 100 0

Table 5. Gradient profile for method 3 [42].


0 95 5

90 55 45

92 10 90

94 10 90

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2.2.2 Saliva

Prior to LC-MS/MS analysis, proteins in saliva samples were first separated with SDS-PAGE using

precast 4-15% Tris-HCl gel. 10 µl of saliva was mixed with 10 µl of reducing sample buffer and

incubated for 5 minutes in 95°C. Then samples were applied to the gel lanes (20 µl per lane). Samples

were run at a voltage of 100V for 75 minutes. Afterwards, proteins were stained with Coomassie blue

and the gel was destained with distilled water.

Selected bands were excised from the gel and in-gel digestion was performed. Firstly, Coomassie

blue staining was removed from gel bands by incubation with 200µl of 0.04M NH4HCO3/50% ACN.

Next, proteins in gel bands were reduced with 100µl of 20mM DTT for 30 minutes in 37°C.

Afterwards, 100µl of 55mM iodoacetamide was added to every sample for alkylation in room

temperature for 20 minutes. Afterwards, proteins were digested with 10µl of 0.02µg/µl of trypsin

solution for 20 minutes on ice and subsequently 20µl of 40mM ammonium bicarbonate/10% ACN

was added and digestion was performed overnight in 37°C. Next day, digested proteins were

extracted from the gel bands with first 60µl ACN for 15 minutes in 37°C and then with 150µl of

50%ACN/5%HCOOH also for 15 minutes at the same temperature. Finally, peptides were dried and

dissolved in 10 µl of water prior to LC-MS/MS analysis and 94 minutes method was used, the same as

for blood samples.

2.2.3 Semen

Semen samples were analyzed similarly to saliva samples described in section 2.2.2. Firstly, proteins

were separated with SDS-PAGE (however, in this case, samples were first diluted 21 times with

phosphate-buffered saline) and later in-gel digestion was performed according to the same protocol.

LC-MS/MS analysis of semen peptides was performed with 20 minutes method (see Table 6).

Table 6. Gradient profile for semen samples.


0 95 5

15 70 30

16 5 95

18 5 95

20 95 5

2.2.4 Fingermarks

For fingermark samples collection, donors (males and females) were asked to put their finger onto a

glass surface, without previous washing their hands, in order to obtain “realistic” conditions.

Deposited fingermarks were later swabbed with cotton swabs. Swabs were subsequently transferred

into Eppendorf vials, 900 µl of 25mM ammonium bicarbonate was added and samples were

incubated at 37°C for 30 minutes. Afterwards, swabs were removed and samples were centrifuged

for 15 minutes at 3000g. The supernatants were collected and in-solution digestion was performed

18

according to the same protocol as for blood samples. For LC-MS/MS analysis, 66 minutes method

was used (see Table 7).

Table 7. Gradient profile for fingermark samples.


0 90 10

60 0 100

65 0 100

65.5 90 10

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3. Results and Discussion

Due to problems with the instrumentation, which had occurred during this research project, not all

samples were analyzed.

This chapter presents the results obtained from analysis of aging body fluids by mass spectrometry.

3.1 Blood

3.1.1 Method optimization

Prior to blood samples analysis, LC-MS/MS conditions were initially optimized with fresh blood

samples. Three different gradients were tested and the number of identified proteins and peptides

was compared. The graphs below present the obtained results.

Figure 4. Number of proteins identified in fresh blood with three different methods.

Figure 5. Number of peptides identified in fresh blood with three different methods.

0

50

100

150

200

Proteins identified

43 minutes method 60 minutes method 94 minutes method

0

200

400

600

800

1000

1200

Peptides identified

43 minutes method 60 minutes method 94 minutes method

20

From the foregoing graphs can be derived that the highest number of identification was obtained by

using 94 minutes method. With this gradient, 179 proteins and 953 peptides were identified. The 60

minutes method provided a significant lower number of proteins and peptide, 117 and 748

respectively. Finally, application of the shortest gradient resulted in 167 identified proteins and 748

identified peptides.

3.1.2 Examining aging of blood

Blood samples were analyzed at three different ages: fresh blood sample (liquid), 1 day old (stain)

and 7 days old blood (stain) with LC-MS/MS. Subsequently, the number of proteins and peptides

from every sample were compared (see Table 8). All measurements were performed in triplicates

and data was generated using human thorough protein database with Protein PilotTM software using

false discovery rate (FDR) 1%.

Table 8. Number of proteins and peptides identified in aged blood samples, in triplicates.

Sample Proteins #1

Proteins #2

Proteins #3

Peptides #1

Peptides #2

Peptides #3

Fresh blood

115 131 139 1280 1712 1646

1 day old 119 130 128 1552 1522 1512

7 days old 129 110 111 1458 1369 1058

Coefficient of variation (CV) in every sample for proteins and peptides, respectively, is as follow: fresh

sample 9.5%, 15%; one day old sample 4.7%, 1.4%; and seven days old sample 9.1%, 16.2%.

In the next step, data was generated using other software. First, Search Gui 2.7.1, and subsequently,

Peptide Shaker 1.8.1. Two combined search engines were used: Open Mass Spectrometry Search

Algorithm (OMSSA) and MS-GF+. The used database for identification was Target-Decoy version

Homo sapiens protein database + common contaminants. Quality control filtering was performed

with 1% FDR. Data from three technical replicates were pooled together.

The number of identified proteins from all three samples was plotted and presented in the Figure 4.

The complete lists of identified proteins in blood samples (using Search Gui and Peptide Shaker) can

be found in Appendices 1-3.

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Figure 6. Comparison of proteins in all three blood samples.

78 proteins were detected in every blood sample. Nonetheless, the number of unique proteins in

every sample is different. These number decreased from 14 proteins in fresh blood to 12 protein in

one day old sample and finally to 4 proteins in 7 days old blood.

Protein quantification was performed by using normalized spectrum abundance factor (NSAF).

10 proteins were found with alterations in NSAF values. 9 proteins have decreasing NSAF value (Fig.

7-15) and for one it is increasing (Fig. 16).

Figure 7. NSAF values for alpha-1 acid glycoprotein-2.

0

0,02

0,04

0,06

0,08

0,1

fresh 1 day old 7 days old

NS

AF

Alpha-1 acid glycoprotein-2

22

Figure 8. NSAF values for alpha-2-macroglobulin.

Figure 9. NSAF values for ankyrin-1.

Figure 10. NSAF values for apolipoprotein B-100.

0

0,005

0,01

0,015

0,02

0,025

0,03

0,035


NS

AF

Alpha-2-macroglobulin

0

0,001

0,002

0,003

0,004

0,005

0,006

0,007


NS

AF

Ankyrin-1

0

0,0005

0,001

0,0015

0,002

0,0025

0,003

0,0035


NS

AF

Apolipoprotein B-100

23

Figure 11. NSAF values for band 3 anion transport protein.

Figure 12. NSAF values for Ig gamma-4 chain region.

Figure 13. NSAF values for haptoglobin.

0

0,01

0,02

0,03

0,04

0,05

0,06

0,07


NS

AF

Band 3 anion transport protein

0

0,01

0,02

0,03

0,04

0,05

0,06


NS

AF

Ig gamma-4 chain C region

0

0,002

0,004

0,006

0,008

0,01


NS

AF

Haptoglobin

24

Figure 14. NSAF values for hemopexin.

Figure 15. NSAF values for putative uncharacterized protein.

Figure 16. NSAF values for putative bisphosphoglycerate mutase.

From all aforementioned proteins, two characterize with significant changes in NSAF values:

apolipoprotein B-100 and haptoglobin. The first one is responsible for lipids transporting and

proteins and phospholipids binding (See Table 9). The latter one is accountable for capturing and

0

0,01

0,02

0,03

0,04

0,05

0,06


NS

AF

Hemopexin

0

0,02

0,04

0,06

0,08

0,1


NS

AF

Putative uncharacterized protein

0,019

0,02

0,021

0,022

0,023

0,024

0,025

0,026


NS

AF

Bisphosphoglycerate mutase

25

combining free plasma hemoglobin and it allows for hepatic cyclin of heme iron which prevents the

damage of kidneys.

For two proteins, alpha-1 acid glycoprotein-2 and putative uncharacterized proteins, the NSAF value

is decreasing notably during first 24 hours, however, posteriorly, this decline is less noticeably.

Alpha-1 acid glycoprotein-2 is transporting protein, however, the function of putative

uncharacterized proteins in humans is still unknown. In other mammals, mouse for example, it has a

regulation function of proteins activity.

Only bisphosphoglycerate mutase protein demonstrates an increasing trend in NSAF values over

examined time period. It can be explained by the function of this protein, to regulate hemoglobin

oxygen affinity, which is stronger with the increasing age of the sample.

Table 9. Functions of proteins with changing NSAF values. Information took from http://www.uniprot.org/.

Protein Function

Alpha-1 acid glycoprotein-2 Transporting protein; binding hydrophobic ligands and synthetic drugs

Alpha-2-macroglobulin Inhibiting proteinases by unique 'trapping' mechanism

Ankyrin-1 Attaching integral membrane proteins to cytoskeletal elements

Apolipoprotein B-100 Lipid transporter activity, binding proteins and phospholipids,

Band 3 anion transport protein Transporting protein, mediator in electroneutral anion exchange across the cell membrane; structural protein

Bisphosphoglycerate mutase regulating hemoglobin oxygen affinity, exhibiting mutase and phosphatase activities

Ig gamma-4 chain C region Antigen binding

Haptoglobin Capturing and combining with free plasma hemoglobin (preventing kidney damage)

Hemopexin Binding heme and transporting it to the liver for breakdown and iron recovery

Putative uncharacterized protein Unknown function in homo sapiens

3.2 Saliva

Saliva samples at ages of one day old, one week old and three weeks old were analyzed.

Firstly, proteins were separated with SDS-PAGE (Fig. 17). The arrows indicate from which part of the

gel bands were excised. Per sample, three bands were excised and each band was analyzed

separately. Afterwards, results from three bands were pooled together.

26

Figure 17. SDS-PAGE of saliva samples.

Data were generated according to the second step with blood analysis, by using First Search Gui 2.7.1

and Peptide Shaker 1.8.1 software. The number of all identified proteins in all three samples is

presented in the Venn diagram below. The all lists of proteins identified in saliva samples can be

found in Appendices 4-6.

Figure 18. Comparison of proteins in all three saliva samples.

39 proteins were present in every saliva sample, nonetheless, the number of unique proteins

detected is increasing with the age of saliva samples, from 9 unique proteins in one day old sample to

22 in three weeks old.

27

Similarly to blood samples, saliva samples also underwent quantification with NSAF. For eight

proteins, an alteration of NSAF was observed (See Fig. 19-26).

Figure 19. NSAF values for keratin, type I cytoskeletal 13.


Figure 21. NSAF values for keratin, type II cytoskeletal 2 epidermal.

0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

0,16

one day one week 3 weeks

NS

AF

Keratin, type I cytoskeletal 13

0

0,005

0,01

0,015

0,02

0,025


NS

AF


0

0,005

0,01

0,015

0,02

0,025


NS

AF

Keratin, type II cytoskeletal 2 epidermal

28

Figure 22. NSAF values for keratin, type II cytoskeletal 3.

Figure 23. NSAF values for keratin, type II cytoskeletal 6A.

Figure 24. NSAF values for lactotransferrin.

0

0,01

0,02

0,03

0,04

0,05


NS

AF

Keratin, type II cytoskeletal 3

0

0,005

0,01

0,015

0,02

0,025

0,03

0,035

0,04


NS

AF

Keratin, type II cytoskeletal 6A

0

0,002

0,004

0,006

0,008

0,01

0,012


NS

AF

Lactotransferrin

29

Figure 25. NSAF values for leukocyte elastase inhibitor.

Figure 26. NSAF values for tubulin alpha-4A chain.

Interestingly, in this case, there are more proteins in which NSAF values were increasing with the age

of the sample. Moreover, most of proteins with variation in NSAF are keratin type proteins. The most

significant changes occur for keratin, type I cytoskeletal 13 with upward trend and for keratin, type II

3 with the downward trend. Furthermore, those two proteins have similar function: structural

molecule activity. The other two proteins with considerable alterations are leukocyte elastase

inhibitor and tubulin alpha-4A chain. The first one is responsible for the activity of neutrophil

proteases elastase, and the latter one for GTPase activity and GTP binding.


Protein Function

Keratin, type I cytoskeletal 13 Structural molecule activity; cellular response to retinoic acid; tongue morphogenesis

Keratin, type I cytoskeletal 17 Structural molecule activity

Keratin, type II cytoskeletal 2 epidermal Associated with keratinocyte activation; proliferation and keratinization

Keratin, type II cytoskeletal 3 Structural molecule activity; intermediate filament cytoskeleton organization

0

0,01

0,02

0,03

0,04

0,05


NS

AF

Leukocyte elastase inhibitor

0

0,005

0,01

0,015

0,02

0,025

0,03

0,035


NS

AF

Tubulin alpha-4A chain

30

Keratin, type II cytoskeletal 6A Involved in the activation of follicular keratinocytes after wounding

Lactotransferrin Iron ion binding

Leukocyte elastase inhibitor Regulating the activity of the neutrophil proteases elastase

Tubulin alpha-4A chain GTPase activity, GTP binding

3.3 Semen

Semen samples (fresh, one week old and two weeks old) were analyzed according to the same

protocol as used for saliva samples. Proteins were first separated with SDS-PAGE, then 7 bands per

lane were excised and analyzed separately. At the end, results were pooled together for every

sample.

Figure 27. SDS-PAGE of semen samples.

31

The Venn diagram below depicts the identified proteins in all semen samples. The lists of all

identified proteins in semen samples can be found in Appendices 7-9.

Figure 28. Comparison of proteins in all three semen samples.

In the case of this body fluid, 41 common proteins for all aging samples were found, however, the

number of the unique proteins is variable. In fresh samples 36 unique proteins were identified. In 7

days old samples this number increased to 37. Nevertheless, this number decreased to only 11

unique proteins in two weeks old samples.

Proteins in all semen samples were quantified with NSAF and 6 proteins showed variations in NSAF

values (Fig. 29-34).

Figure 29. NSAF values for clusterin.

0

0,005

0,01

0,015

0,02

0,025

fresh one week two weeks

NS

AF

Clusterin

32

Figure 30. NSAF values for dermcidin.


Figure 32. NSAF values for keratin, type II cytoskeletal 6A.

0

0,05

0,1

0,15

0,2


NS

AF

Dermcidin

0

0,01

0,02

0,03

0,04

0,05

0,06


NS

AF


0

0,002

0,004

0,006

0,008

0,01

0,012


NS

AF

Keratin, type II cytoskeletal 6A

33

Figure 33. NSAF values for semenogelin-2.

Figure 34. NSAF values for serum albumin.

Keratin proteins are also present in semen samples with the downward or upward trend for type I

and type II, respectively. However, there are two proteins which NSAF is changing significantly. First

one, dermcidin, with upward trend, has antimicrobial properties. The second one, with reducing

NSAF, is semenogelin-2, which is involved in gel matrix formation. This result can indicate why the

older semen is more liquid then the fresh one.


Protein Function

Clusterin Maintains partially unfolded proteins in a state appropriate for subsequent refolding by other chaperones

Dermcidin

Displays antimicrobial activity; exhibits proteolytic activity


Cellular response to calcium ion

Keratin, type II cytoskeletal 6A Involved in the activation of follicular keratinocytes after wounding

Semenogelin-2

Participates in the formation of a gel matrix

0

0,005

0,01

0,015

0,02

0,025


NS

AF

Semenogelin-2

0

0,005

0,01

0,015

0,02

0,025

0,03

0,035


NS

AF

Serum albumin

34

Serum albumin

Transporting

3.4 Fingermarks

7 female (2 donors) and 6 male (one donor) fingermarks samples were analyzed at different age:

fresh (only female), one day old, 10 days old and 17 days old. Results were generated with Protein

Pilot software. Table 12 presents proteins identified in each sample.

Table 12. Proteins identified in every fingermark sample.

Sample Proteins

Fresh female fingermark keratin, type I cytoskeletal (9, 10, 14, 16); keratin, type II cytoskeletal (1, 1b, 2 epidermal, 5, 6A, 78); dermcidin; keratinocyte proline-rich protein; filaggrin-2; skin-specific protein 32; loricrin; junction plakoglobin

One day old female fingermark keratin, type II cytoskeletal 1

One day old male fingermark keratin, type I cytoskeletal (9, 10, 13, 14, 15, 16, 17); keratin, type II cytoskeletal (1, 2 epidermal); charged multivesicular body protein 2a; serum albumin; phosphofurin acidic cluster sorting protein 1

10 days old female fingermark Keratin, type I cytoskeletal 9; keratin, type II cytoskeletal (1, 2 epidermal, 5, 6A, 73); apoptosis-enhancing nuclease;

10 days old male fingermark Keratin, type I cytoskeletal 9; keratin, type II cytoskeletal (1, 2 epidermal); sodium/potassium-transporting ATPase subunit alpha-4; dermcidin

17 days old female fingermark Keratin, type I cytoskeletal (9, 10, 14, 16, 17); keratin, type II cytoskeletal (1, 2 epidermal, 5); dermcidin

17 days old male fingermark Keratin, type I cytoskeletal 10; prolactin-inducible protein

All samples contain keratin proteins, which is a protein found in skin cells. Interestingly, some

samples contain proteins which were not found in other fingermarks. This can suggest that those

proteins originate from contamination, as the donors deposited their fingermarks directly on the

glass slides, without previous finger preparation.

The results from fingermark samples could not be quantified as the signal to noise ratio in the

spectra was too low.

35

4. Conclusions and future research

For the first time mass spectrometry method was applied to study the aging of blood, saliva, semen

and fingerprints. In this research, protein changes were investigated, which occurs when the body

fluid is aging. It was shown that for some proteins, their concentration is increasing with time, for

example biphosphoglycerate mutase in blood, or keratins, type II in saliva. There were also several

proteins with decreasing concentration, such as apolipoprotein B-100 or semengelin-2. In the present

study, it was observed that for saliva, more proteins increased in concentration in comparison to

blood and semen. With regard to fingermarks samples, in this case, different extraction method is

required to obtain enough protein concentration that can be measured. This work presented the

potential of mass spectrometry technique for age determination of four body fluids commonly found

on a crime scene, although more research is needed in this area.

For future research, it is recommended to broaden the range of analyzed samples. In the case of

investigating proper biomarkers, it is important to expand studies to several donors from both sexes

with a wider range of age. Additionally, in forensic science it is significant to investigate fluids on

different substrates. Also, environmental conditions are crucial, for example how proteins undergo

degradation at different temperatures and humidity levels. Furthermore, label-free quantification

methods used in this study, can also suggest that more accurate methods should be applied.

36

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39

The data presented in appendixes were generated by Search Gui 2.7.1 and Peptide Shaker 1.8.1.

Note that some proteins are identified more than one time per sample. emPAI - exponentially

modified protein abundance index; NSAF - normalized spectrum abundance factor; MW – molecular

weight.

Appendix 1. All validated proteins in fresh blood sample.

Accession

Protein Inference

Class

Protein name

Sequence Coverage

(%)

#Validated

Peptides

emPAI

NSAF

MW (kDa)

P01024 Unrelated Proteins

Complement C3

40.88995790739627

44 0.714125253

2913134 0.01152003971

2574301 187.0298753

1966258

F6KPG5 Single

Protein Albumin

(Fragment) 76.7521367

5213675 56

4.115178099293143

0.0685492934053836

66.48798147102801

P01023 Single

Protein Alpha-2-

macroglobulin 47.354138398914515

44 1.249054605

8357808 0.02915375633

5619455 163.1878914

6924135

P11277

Related and

Unrelated Proteins

Spectrin beta chain,

erythrocytic

17.501169864295743

26 0.249264143

7899512 0.00369787553

21113903 246.3157334

321971

A0A087WZE4

Related Proteins

Spectrin alpha chain,

erythrocytic 1

15.720164609053498

23 0.177572498

21932586 0.00339278233

15763428 280.9387841

720546

A0A087WTM7

Related and

Unrelated Proteins


6.883057090239411

18 0.091578487

87739664 0.00315395313

4692732 489.5273949

3234196

C8C504 Single

Protein Beta-globin

95.91836734693877

23 33.14548873

833603 1.08852418504

30756 15.98734254

8192922

P02730

Related and

Unrelated Proteins


24.259055982436884

15 0.674482438

0115348 0.05793226863

3375625 101.7274121

8512044

P02042 Related Proteins

Hemoglobin subunit delta

93.19727891156462

18 14.84893192

4611133 0.45956003636

3566 16.04528986

0997062

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

21 0.790655529

6923772 0.15593423608

704202 69.18136901

532891

A0A0K0K1H8

Related and

Unrelated Proteins

Epididymis secretory

sperm binding protein Li 71p

33.3810888252149

19 0.739828052

9303642 0.04192341630

458897 77.02963611

452142

P02787

Related and

Unrelated Proteins

Serotransferrin

32.5214899713467

19 0.739828052

9303642 0.02566325370

2962904 77.01362996

651181

P02787

Related and

Unrelated Proteins

Serotransferrin

32.95128939828081

19 0.739828052

9303642 0.02241122118

2637716 77.01362996

651181

P02671 Single

Protein Fibrinogen alpha chain

28.521939953810627

17 0.552427173

4347945 0.02193464511

4181275 94.91441497

589778


Catalase 35.863377609108156

13 0.690714103

4735806 0.03718063723

218359 59.71875531

355211

P16157 Single

Protein Ankyrin-1

13.131313131313131

15 0.207722655

13446264 0.00599593074

032388 206.1369244

395263


Fibrinogen beta chain

50.30549898167006

16 0.887391822

1350972 0.04582730151

411469 55.89226078

115152

A0A0G2JL Related Complement 12.0141342 12 0.174268583 0.00282639279 187.5979404

40

54 Proteins C4-B 75618375 45092833 24914566 9222188

I1VZV6 Single

Protein Hemoglobin

alpha 1 92.9577464

7887323 15

13.251026703029977

2.749347740393739

15.269957551186833


Apolipoprotein A-I

55.430711610486895

17 1.801356761

1988681 0.15414244973

89475 30.75893250

0112536

Q3LR79 Single

Protein Hemoglobin

beta 93.3333333

3333333 15

30.622776601683793

1.0646784733306542

11.474842789202496

A0A024R6I7

Related Proteins

Alpha-1-antitrypsin

40.19138755980861

13 1.198392648

8622894 0.13958117324

59462 46.67898813

8410034

Q9BWU5 Single

Protein

Mutant hemoglobin beta chain (Fragment)

85.71428571428571

13 14.19911082

9529339 1.16773237162

46961 11.49394786

3407726

D9YZU8 Single

Protein Hemoglobin,

gamma A 85.7142857

1428571 12

5.309573444801933

0.24368578322660492

16.11827504126487

C9JC84 Related Proteins

Fibrinogen gamma chain

33.839479392624725

10 0.687612475

7881477 0.05015322108

597598 52.30432646

809949

P00918

Related and

Unrelated Proteins

Carbonic anhydrase 2

48.07692307692308

9 1.154434690

0318838 0.08711270534

414638 29.22792218

6630536

Q4TZM4 Single

Protein

Hemoglobin beta chain (Fragment)

88.11881188118812

12 14.84893192

4611133 1.94561481731

5928 11.01563886

7792866


Vitamin D-binding protein

36.91983122362869

9 0.429461338

35684486 0.01950723327

3056056 52.92902967

385641

P02790 Single

Protein Hemopexin

36.58008658008658

11 0.778279410

0389228 0.05063651626

151628 51.64327297

890149

A0A075B6N9

Related Proteins

Ig mu chain C region

(Fragment)

31.346578366445915

11 0.947483039

9087562 0.03887085137

085134 49.40858023

156175


Peroxiredoxin-6

62.05357142857142

10 1.154434690

0318838 0.04061968537

4149665 25.01919029

056794

P06727 Single

Protein Apolipoprotei

n A-IV 15.151515151515152

5 0.242623671

9115049 0.01489071038

2513661 45.37146664

308862


L-lactate dehydrogenas

e B chain

29.34131736526946

7 0.606506003

8537282 0.01930001682

6518603 36.61514296

895986

A0A087WU08

Related Proteins

Haptoglobin 39.8576512

455516 9

0.9952623149688795

0.035842008868324615

31.3878684863898



2.4326101249178174

7 0.033029926

90742457 0.00166372245

1468622 515.2829109

17923

B7ZKJ8

Related and

Unrelated Proteins

ITIH4 protein 8.55614973

262032 5

0.13977251980662686

0.006619537275064268

103.81622401008842

P11277

Related and

Unrelated Proteins


erythrocytic

3.0416471689284044

5 0.043728062944320634

0.0013099547156802832

246.3157334321971

P32119

Related and

Unrelated Proteins

Peroxiredoxin-2

49.494949494949495

10 1.993577294

7204895 0.16687101854

784764 21.87823782

783635

Q6MZU6

Related and

Unrelated Proteins

Putative uncharacteriz

ed protein DKFZp686C15

213

27.586206896551726

9 0.701254279

8525889 0.07749920340

380885 51.06619379

122771

A0A075B6N8

Related and


34.48275862068966

9 0.750827031

7357244 0.02486747170

9576934 41.29932388

898452

41

Unrelated Proteins

(Fragment)

O95408 Single

Protein Beta globin (Fragment)

71.9298245614035

8 99.0 1.61262328095

1787 6.262154763

39843

Q6J1Z9 Single

Protein

Hemoglobin alpha 1

(Fragment) 90.0 9 9.0

1.2764004083448635

9.585181362001366

Q53GY3

Related and

Unrelated Proteins

Angiotensinogen variant (Fragment)

25.56701030927835

8 0.692666615

037876 0.03076587301

587301 53.05051576

500968

E9PFZ2

Related and

Unrelated Proteins

Ceruloplasmin 8.87949260

0422834 5

0.1274137659327852

0.003986810551558753

108.75141618854433


Purine nucleoside

phosphorylase

35.6401384083045

6 0.637893706

954064 0.02866205305

6516718 32.09715605

245075

A0A087WV47

Related and

Unrelated Proteins


25.10729613733906

8 0.567191080

3783076 0.10509241139

378137 51.12147771

750289


Flavin reductase (NADPH)

50.0 7 1.335721469

0901222 0.14358529819

694865 22.10542642

5215388

P01876

Related and

Unrelated Proteins

Ig alpha-1 chain C region

32.29461756373937

8 1.310129700

0831598 0.03864066547

120943 37.63063836

9583146

P01876

Related and

Unrelated Proteins


36.26062322946176

8 1.310129700

0831598 0.05537706714

484954 37.63063836

9583146


Antithrombin-III

15.948275862068964

5 0.228247006

34692308 0.01217672413

7931035 52.56886431

029427



24.324324324324326

4 0.321941148

46602906 0.02123552123

552123 29.98651036

8450627


Kininogen-1 7.91925465

8385093 3

0.10529514112602167

0.009451795841209828

71.91215085210732

E9PFZ2

Related and

Unrelated Proteins


5 0.127413765

9327852 0.00879296562

7498003 108.7514161

8854433

Q86YQ1 Single

Protein

Hemoglobin alpha-2

(Fragment)

87.91208791208791

6 4.623413251

903491 0.71365976578

52747 9.665182424

630526

E5RFE7 Related Proteins

Carbonic anhydrase 1 (Fragment)

31.443298969072163

7 1.448436746

8222272 0.18008751710

050394 21.38873696

621423

A0A0G2JPD4

Related and

Unrelated Proteins


(Fragment)

20.489296636085626

6 0.637893706

954064 0.05183673469

3877486 35.91786587

959916

A0A087WT59

Related Proteins

Transthyretin 38.3783783

7837838 5

1.2758459260747883

0.09604978354978352

20.132907042394372

P02787

Related and

Unrelated Proteins

Serotransferrin

9.025787965616045

6 0.191103133

28300688 0.03028455284

5528463 77.01362996

651181

A0A0C4D Related Haptoglobin 8.64553314 4 0.311133937 0.01055194805 38.42734787

42

GL8 Proteins 1210375 42156426 1948054 385203

G3V3A0

Related and

Unrelated Proteins

Alpha-1-antichymotry

psin

16.097560975609756

3 0.389495494

3731377 0.03170731707

317073 23.39110667

577842


Triosephosphate isomerase

22.377622377622377

3 0.258925411

7941673 0.01923076923

076923 30.77169610

1193438

H0Y7Z1

Related and

Unrelated Proteins

Fibronectin (Fragment)

5.530371713508613

3 0.086787359

11863655 0.00180188504

89743114 120.6992441

0608232

Q86YQ4 Single

Protein

Alpha-1 globin

(Fragment)

90.9090909090909

5 3.216965034

2858223 0.77914141414

14315 9.476096235

395074

E5RFL2 Related Proteins


60.16949152542372

6 3.641588833

6127784 0.43644067796

61018 13.24825410

0518784

P02787

Related and

Unrelated Proteins

Serotransferrin

6.59025787965616

4 0.123654800

13875096 0.03523035230

352302 77.01362996

651181

P19652 Single

Protein Alpha-1-acid

glycoprotein 2 27.363184079601986

5 0.687612475

7881477 0.07718375693

835813 23.58763169

0019066


Alpha-1-acid glycoprotein 1

33.33333333333333

6 0.930697728

8832501 0.06315513626

834378 23.49675931

597699


Delta-aminolevulini

c acid dehydratase

22.12121212121212

5 0.417474162

9268053 0.02615384615

3846156 36.27141416

930379

P08603

Related and

Unrelated Proteins

Complement factor H

2.5995125913891144

2 0.037225095

40705721 0.00195694716

24266144 139.0046981

6836323

M0R009 Related Proteins

Alpha-1B-glycoprotein (Fragment)

10.130718954248366

2 0.185971012

33766966 0.00837520938

0234505 33.43379931

857088


Protein 4.1 4.16666666

6666666 2

0.041223256048305235

0.003546099290780142

96.95728291972311

B2M1S7 Single

Protein

Beta-globin Showa

Yakushiji variant

(Fragment)

100.0 4 20.54434690

0318832 1.70899470899

47093 4.493394684

7851916

A0A075B6H6

Related and

Unrelated Proteins

Ig kappa chain C region

(Fragment)

79.43925233644859

5 1.848035868

4358014 0.47472185135

7368 11.71166718

8377447

A0A087WV45

Related Proteins


9064749 4

1.0309176209047357

0.13237410071942446

15.110588167739953

A0A087WU08

Related Proteins


1708185 4

0.35935639087852556

0.01361445571971888

31.3878684863898

E9PGN7 Related Proteins

Plasma protease C1

inhibitor

9.392265193370166

4 0.216483949

025851 0.00699440447

64188645 59.45455599

2296385

G3V595

Related and

Unrelated Proteins


psin (Fragment)

15.081967213114755

3 0.258925411

7941673 0.01498127340

82397 34.60356994

9471684

Q13228 Related Proteins

Selenium-binding

protein 1

10.16949152542373

3 0.162030136

3862613 0.01059322033

8983049 52.35762502

132521

P02749 Related Beta-2- 19.4202898 4 0.311133937 0.01135265700 38.27265933

43

Proteins glycoprotein 1 55072465 42156426 4830919 588615


Alpha-1B-glycoprotein

8.282828282828284

2 0.118872211

58742212 0.01318944844

1247004 54.21953944

303767



29.381443298969074

4 0.668100537

2000588 0.17207792207

792205 21.38873696

621423



15.979381443298967

4 0.668100537

2000588 0.17911255411

255414 21.38873696

621423

A0A0B4J231

Related and

Unrelated Proteins

Immunoglobulin lambda-

like polypeptide 5

32.093023255813954

4 0.623776739

1887217 0.06910112359

550558 23.13567037

3513683

C9JVG0 Related Proteins

Serotransferrin (Fragment)

26.865671641791046

4 1.030917620

9047357 0.13258426966

29213 14.68097224

2798342

E7EUT5 Related Proteins

Glyceraldehyde-3-

phosphate dehydrogenas

e

25.76923076923077

4 0.425102670

3029981 0.05743243243

243244 27.85306080

5444024

A0A0A0MRQ5

Related and

Unrelated Proteins

Peroxiredoxin-1

29.896907216494846

3 0.873817422

8603839 0.04786450662

739325 10.66950712

5347321

C9JV77 Related Proteins

Alpha-2-HS-glycoprotein

21.467391304347828

4 0.389495494

3731377 0.03721374045

801526 39.38672460

1822


Apolipoprotein A-II

19.0 3 0.778279410

0389228 0.065

11.16789735900159

H0YAS8 Related Proteins

Clusterin (Fragment)

28.57142857142857

2 0.258925411

7941673 0.05

16.166165820637204

E9PIT3 Related Proteins

Prothrombin 8.91938250

4288166 3

0.119902793272475

0.003569211669770329

65.36674524764112


Inter-alpha-trypsin

inhibitor heavy chain

H1

6.0373216245883645

3 0.082636733

87405435 0.00224791265

25369302 101.3256149

1857365

A0A024R6I7

Related and

Unrelated Proteins

Alpha-1-antitrypsin

10.76555023923445

3 0.199353946

2092342 0.10890151515

151522 46.67898813

8410034


Apolipoprotein A-II

28.999999999999996

2 0.467799267

62206947 0.36666666666

666686 11.16789735

900159

P60709

Related and

Unrelated Proteins

Actin, cytoplasmic 1

17.333333333333336

3 0.211527658

62858836 0.022

41.709730139029176


Alpha-1-antitrypsin

5.980861244019139

2 0.128837891

68468907 0.03219696969

69697 46.70701943

8538325

H0Y300 Related Proteins


1764707 3

0.16998910658906596

0.008426183844011143

49.07441753459848

E7EPV7 Related Proteins

Alpha-synuclein

31.30434782608696

2 0.359356390

87852556 0.06956521739

130435 11.76924943

3169955


Plasma protease C1

inhibitor

4.972375690607735

2 0.102943311

79161298 0.00419664268

5851319 59.45455599

2296385

A0A087WTE1

Related Proteins

Inter-alpha-trypsin


H2

4.338624338624339

3 0.073030940

52615622 0.00522252497

729337 106.5897031

5928195

P05109 Single Protein S100- 23.6559139 2 0.389495494 0.04838709677 10.82764922

44

Protein A8 78494624 3731377 419355 1794548

A2NYQ9 Single

Protein

Anti-folate binding protein

(Fragment)

31.666666666666664

3 0.778279410

0389228 0.01342592592

5925928 13.27443279

9882204


Prothrombin (Fragment)

16.358024691358025

3 0.291549665

0148839 0.01378896882

4940049 35.90878157

243862


Transitional endoplasmic

reticulum ATPase

4.094292803970223

2 0.048113134154685655

0.004267425320056899

89.26571926128344

B7UCU6

Related and

Unrelated Proteins

Gamma-globin chain (Fragment)

19.047619047619047

2 0.519911082

9529336 0.64588744588

74546 11.57887621

4589096

A0A087WV47

Related and

Unrelated Proteins


6.866952789699571

2 0.118872211

58742212 0.03789954337

8995426 51.12147771

750289

A0A075B6P5

Related and

Unrelated Proteins

Protein IGKV2-28

(Fragment)

30.833333333333336

2 1.154434690

0318838 0.18604651162

790695 12.94845774

1318867


Solute carrier family 2,

facilitated glucose

transporter member 1

4.67479674796748

2 0.136463666

38572482 0.02124183006

5359478 54.04853579

695947

A0A0C4DH32

Related and

Unrelated Proteins

Protein IGHV3-20

(Fragment)

25.64102564102564

2 0.668100537

2000588 0.01282051282

0512822 12.70929366

8088954

B4DJ51

Related and

Unrelated Proteins

Calmodulin 1 (Phosphorylas

e kinase, delta),

isoform CRA_a

20.13422818791946

2 0.425102670

3029981 0.02232142857

1428572 16.82683430

0721597

C9JF17 Related Proteins

Apolipoprotein D

(Fragment)

10.232558139534884

2 0.311133937

42156426 0.01162790697

6744186 24.14216018

7180192

A0A0C4DGV7

Related Proteins

Retinol-binding

protein 4

14.572864321608039

2 0.202264434

61741295 0.01005025125

6281407 22.85315160

2540063



15.979381443298967

4 0.668100537

2000588 0.17911255411

255414 21.38873696

621423

Appendix 2. All validated proteins in one day old blood sample.

Accession

Protein Inference

Class

Protein name Sequence

Coverage (%)

#Validated Peptides

emPAI

NSAF

MW (kDa)


Complement C3

42.3331328923632

48 0.8001933328915447

0.014698652477377191

187.02987531966258

P01023 Single

Protein Alpha-2-


8602446 44

1.2490546058357808

0.0229620407409577

163.18789146924135

A0A087WZE4

Related Proteins


erythrocytic 1

14.362139917695472

22 0.1692334586063975

0.004608800035677576

280.9387841720546

45

P11277

Related and

Unrelated Proteins


erythrocytic

14.225549836218999

21 0.1969249348971422

5

0.004950891467032518

246.3157334321971

C8C504 Single

Protein Beta-globin

95.91836734693877

23 33.14548873833603

1.29374193690661

15.987342548192922

A0A087WTM7

Related and

Unrelated Proteins


3.4069981583793743

9 0.0447863359928655

2

0.0025306842333036477

489.52739493234196

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

22 0.8410273525621605

0.05517801097802998

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

33.443708609271525

22 0.8410273525621605

0.04338165336213569

69.18136901532891



93.19727891156462

18 14.848931924611133

0.5795009151570016

16.045289860997062

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

21 0.7906555296923772

0.14441180197941006

69.18136901532891


Apolipoprotein A-I

55.430711610486895

18 1.976351441631318

0.1752561778751475

30.758932500112536

A0A0K0K1H8

Related and

Unrelated Proteins



30.945558739255013

19 0.7398280529303642

0.045122985368780594

77.02963611452142

P02787

Related and

Unrelated Proteins

Serotransferrin

30.08595988538682

19 0.7398280529303642

0.028862822767154534

77.01362996651181

P02730

Related and

Unrelated Proteins


20.417124039517013

12 0.5104434757204741

0.04518471483416132

101.72741218512044

P02671 Single


23.556581986143186

16 0.5127782816797017

0.02231942486851851

94.91441497589778

Q3LR79 Single

Protein

Hemoglobin beta

(Fragment)

95.23809523809523

16 38.810717055349734

1.347167243856381

11.474842789202496

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

30.629139072847682

19 0.6940088022878714

0.03932995605270707

69.18136901532891



45.010183299389006

15 0.8139306939110624

0.0424301339685955

55.89226078115152

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

40.909090909090914

17 1.1877616239495525

0.11331533960494844

45.11835747484806

P16157 Single

Protein Ankyrin-1

8.452950558213717

11 0.1484425891565204

2

0.005237519370105122

206.1369244395263

Q9BWU5 Single

Protein


87.61904761904762

14 17.73817422860384

1.3338945330661254

11.493947863407726

I1VZV6 Single

Protein Hemoglobin

alpha 1 85.21126760

56338 15

13.251026703029977

2.006994337243872

15.269957551186833

46

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

39.39393939393939

17 1.1877616239495525

0.05791823516680009

45.11835747484806


Catalase 33.77609108

1593925 12

0.6237767391887217

0.04250658276431473

59.71875531355211

Q4TZM4 Single

Protein


90.0990099009901

13 18.952623149688797

2.304581351004367

11.015638867792866

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

29.545454545454547

17 1.1877616239495525

0.13331887535040376

45.11835747484806

D9YZU8 Single

Protein Hemoglobin,

gamma A 89.11564625

850339 15 9.0

0.25476829053070255

16.11827504126487

A0A024R6I7

Related Proteins

Alpha-1-antitrypsin

40.19138755980861

13 1.1983926488622894

0.10672025260093485

46.678988138410034

P00918

Related and

Unrelated Proteins


60.38461538461538

11 1.5550970903525068

0.0862691166184616

29.227922186630536



32.97180043383948

10 0.6876124757881477

0.044682023206968985

52.30432646809949

A0A0G2JL54

Related Proteins

Complement C4-B

7.302709069493522

7 0.0982410543092073

8

0.006079709370659259

187.59794049222188


Peroxiredoxin-6

51.78571428571429

9 0.9952623149688795

0.06390128968253962

25.01919029056794

D6RF35

Related and

Unrelated Proteins


30.252100840336134

8 0.3894954943731377

0.03878301320528211

52.98591682893214

P02790 Single

Protein Hemopexin

28.78787878787879

8 0.5199110829529336

0.04157795329670329

51.64327297890149

A0A075B6N9

Related Proteins


(Fragment)

27.81456953642384

10 0.8329807108324359

0.03578195247270388

49.40858023156175

P06727 Single


n A-IV 11.86868686

8686869 4

0.1897937907435858

2

0.017076502732240435

45.37146664308862

O95408 Single


71.9298245614035

8 99.0 2.07525161818

78904 6.262154763

39843

A0A087WU08

Related Proteins


1672595 10

1.1544346900318838

0.040224712821190244

31.3878684863898

E9PFZ2

Related and

Unrelated Proteins


0528541 7

0.1828145730152692

7

0.003538597693273953

108.75141618854433

P32119

Related and

Unrelated Proteins

Peroxiredoxin-2

49.494949494949495

10 1.9935772947204895

0.1820579488262413

21.87823782783635



45.14563106796117

6 1.0691380811147897

0.14466019417475726

22.105426425215388



1.9066403681788298

5 0.0234830276008564

1

0.0013677113604174149

515.282910917923

Q6J1Z9 Single

Protein

Hemoglobin alpha 1

(Fragment) 90.0 9 9.0

1.2347300461555972

9.585181362001366

Q86YL2 Single Hemoglobin 92.04545454 9 12.335214 0.80824663811 9.486065329

47

Protein alpha-1 chain (Fragment)

545455 32163324 36691 189616

Q9NQT3 Single

Protein

Alpha one globin

(Fragment)

89.01098901098901

9 12.33521432163324

0.7706121335604688

9.621118486961286

Q6MZU6

Related and

Unrelated Proteins



213

27.586206896551722

9 0.7012542798525889

0.056723493490963686

51.06619379122771

A0A075B6N8

Related and

Unrelated Proteins


(Fragment)

34.48275862068966

9 0.7508270317357244

0.02246440428258608

41.29932388898452


Antithrombin-III

18.96551724137931

6 0.2798022139979537

0.012043308702791466

52.56886431029427



e B chain

20.35928143712575

5 0.4030037231905736

5

0.021908127208480566

36.61514296895986

B7ZKJ8

Related and

Unrelated Proteins


224599 5

0.1397725198066268

6

0.006931692985677563

103.81622401008842

A0A087WV47

Related and

Unrelated Proteins


25.10729613733906

8 0.5671910803783076

0.040808139069134004

51.12147771750289

P01876

Related and

Unrelated Proteins


28.89518413597734

7 1.0805675382171707

0.037108399845293766

37.630638369583146

P01876

Related and

Unrelated Proteins


32.86118980169972

7 1.0805675382171707

0.02099582193724452

37.630638369583146


Angiotensinogen

15.463917525773196

5 0.3894954943731377

0.042444444444444444

53.12051471946386


Purine nucleoside

phosphorylase

35.98615916955018

6 0.637893706954064

0.01995386389850058

32.09715605245075


Selenium-binding

protein 1

15.889830508474576

5 0.2843862635897427

0.011899717514124292

52.35762502132521

Q86YQ1 Single

Protein

Hemoglobin alpha-2

(Fragment)

89.01098901098901

7 6.498942093324558

0.6348016129528891

9.665182424630526

Q9HAR8 Single

Protein

Mutant beta-globin

(Fragment) 100.0 5

45.4158883361278

1.6079404466501144

3.2907445311221695

Q6P5S8 Single

Protein IGK@ protein

42.79661016949153

6 1.0691380811147897

0.208164983164983

25.75683450608628



31.443298969072163

7 1.4484367468222272

0.18123535915743688

21.38873696621423

A0A087WT59

Related Proteins


837838 5

1.2758459260747883

0.14069264069264076

20.132907042394372

P02787

Related and

Unrelated Proteins

Serotransferrin

9.025787965616045

5 0.1568875283162822

8

0.030758807588075893

77.01362996651181

P07738 Unrelated Bisphosphogl 11.58301158 2 0.1497569 0.02316602316 29.98651036

48

Proteins ycerate mutase

3011583 953977358 6023165 8450627

G3V3A0

Related and

Unrelated Proteins


psin

14.634146341463413

3 0.3894954943731377

0.03414634146341463

23.39110667577842

P11277

Related and

Unrelated Proteins


erythrocytic

2.339728591483388

4 0.0348320919317066

66

0.0013961359469750387

246.3157334321971



12.237762237762238

2 0.1659144011798317

0.008741258741258742

30.771696101193438

Q86YQ4 Single

Protein

Alpha-1 globin

(Fragment)

90.9090909090909

5 3.2169650342858223

0.7371921390013633

9.476096235395074



60.16949152542372

6 3.6415888336127784

0.46892655367231645

13.248254100518784

A0A0G2JPD4

Related and

Unrelated Proteins


(Fragment)

20.489296636085626

6 0.637893706954064

0.04174777603349031

35.91786587959916



33.33333333333333

6 0.9306977288832501

0.05607966457023061

23.49675931597699

A0A0C4DGL8

Related Proteins


008646 3

0.2252798573828647

0.013257575757575761

38.42734787385203


Delta-aminolevulini

c acid dehydratase

22.424242424242426

5 0.4174741629268053

0.02878205128205128

36.27141416930379

P19652 Single


glycoprotein 2 28.85572139

3034827 5

0.6876124757881477

0.06129126497224654

23.587631690019066


Kininogen-1 5.124223602

484472 2

0.0690192046030009

3

0.012287334593572778

71.91215085210732

P08603

Related and

Unrelated Proteins

Complement factor H

3.4930950446791225

3 0.0563541037491872

57

0.003180039138943248

139.00469816836323

B2M1S7 Single

Protein

Beta-globin Showa

Yakushiji variant

(Fragment)

100.0 5 45.4158883361278

1.4656771799628925

4.4933946847851916

A0A087WV45

Related Proteins


064749 4

1.0309176209047357

0.12826310380267214

15.110588167739953

P02787

Related and

Unrelated Proteins

Serotransferrin

6.59025787965616

4 0.1236548001387509

6

0.03455284552845528

77.01362996651181

A0A087WU08

Related Proteins


708185 4

0.3593563908785255

6

0.017666543982333454

31.3878684863898


Glyceraldehyde-3-


e

25.769230769230766

4 0.4251026703029981

0.0581831831831832

27.853060805444024



24.184782608695652

5 0.5085907086001784

0.05133587786259543

39.386724601822


Plasma protease C1

9.208103130755065

4 0.216483949025851

0.016986410871302957

59.454555992296385

49

inhibitor

E9PFZ2

Related and

Unrelated Proteins


372093 4

0.1006941712522095

9

0.007793764988009592

108.75141618854433

P00352

Related and

Unrelated Proteins

Retinal dehydrogenas

e 1

7.984031936127744

3 0.1364636663857248

2

0.009830205540661306

54.82694780360638


Phosphoglycerate kinase 1

8.393285371702639

2 0.0944997993195435

5

0.008393285371702638

44.58612676613191



29.381443298969074

4 0.6681005372000588

0.1753246753246753

21.38873696621423



15.979381443298967

4 0.6681005372000588

0.19832251082251087

21.38873696621423



26.865671641791046

4 1.0309176209047357

0.13772070626003205

14.680972242798342

A0A0A0MRQ5

Related and

Unrelated Proteins

Peroxiredoxin-1

29.896907216494846

3 0.8738174228603839

0.046907216494845375

10.669507125347321


Apolipoprotein A-II

19.0 3 0.7782794100389228

0.05 11.16789735

900159



28.57142857142857

2 0.2589254117941673

0.04642857142857143

16.166165820637204


Inter-alpha-trypsin


H1

5.26893523600439

3 0.0826367338740543

5

0.006743737957610789

101.32561491857365

B7ZKJ8

Related and

Unrelated Proteins


31016 3

0.0816603471464902

9

0.004627249357326479

103.81622401008842



288166 3

0.119902793272475

0.0026381129733085043

65.36674524764112


Plasminogen 4.814814814

814815 2

0.0556729942333293

9

0.003536067892503536

90.5101571496777

A0A024R6I7

Related and

Unrelated Proteins

Alpha-1-antitrypsin

10.76555023923445

3 0.1993539462092342

0.09469696969696971

46.678988138410034


Apolipoprotein A-II

28.999999999999996

2 0.4677992676220694

7 0.33

11.16789735900159

C9J5S7 Related Proteins

Peptidyl-prolyl cis-

trans isomerase

26.446280991735538

3 0.7782794100389228

0.07107438016528922

13.199402292131417



trans isomerase A

20.606060606060606

3 0.4125375446227544

0.02787878787878789

18.000885276520645



764707 3

0.1699891065890659

6

0.006963788300835654

49.07441753459848

K7ERI9 Related Proteins

Apolipoprotein C-I

28.57142857142857

3 0.8738174228603839

0.15503875968992248

8.641669993226936

50

(Fragment)

A0A087WTE1

Related Proteins

Inter-alpha-trypsin


H2

2.751322751322751

2 0.0481131341546856

55

0.004768392370572207

106.58970315928195

B0YIW2 Related Proteins

Apolipoprotein C-III

29.059829059829063

2 0.4677992676220694

7

0.05555555555555555

12.80745671066277


Prothrombin (Fragment)

10.802469135802468

2 0.1859710123376696

6

0.008992805755395683

35.90878157243862

B7UCU6

Related and

Unrelated Proteins


19.047619047619047

2 0.5199110829529336

0.6742857142857167

11.578876214589096

A0A087WV47

Related and

Unrelated Proteins


6.866952789699571

2 0.1188722115874221

2

0.037964774951076315

51.12147771750289

A0A0B4J231

Related and

Unrelated Proteins


like polypeptide 5

13.023255813953488

2 0.2742749857031337

0.05243445692883893

23.135670373513683

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

2.525252525252525

2 0.0964781961431850

9

0.011363636363636364

45.11835747484806


Alpha-1-antitrypsin

5.980861244019139

2 0.1288378916846890

7

0.027462121212121215

46.707019438538325


Alpha-synuclein

31.30434782608696

2 0.3593563908785255

6

0.0782608695652174

11.769249433169955

A0A075B6P5

Related and

Unrelated Proteins

Protein IGKV2-28

(Fragment)

30.833333333333336

2 1.1544346900318838

0.16279069767441862

12.948457741318867



facilitated glucose


4.67479674796748

2 0.1364636663857248

2

0.021241830065359478

54.04853579695947

A0A087WTE1

Related Proteins

Inter-alpha-trypsin


H2

3.1746031746031744

2 0.0481131341546856

55

0.004768392370572207

106.58970315928195

P05109 Single

Protein Protein S100-

A8 23.65591397

8494624 2

0.3894954943731377

0.04838709677419355

10.827649221794548

J3QRN2 Related Proteins

Beta-2-glycoprotein 1

(Fragment)

20.441988950276244

2 0.4677992676220694

7

0.019337016574585635

19.893845899589213

A0A087WV47

Related and

Unrelated Proteins


4.291845493562231

2 0.1188722115874221

2

0.00547945205479452

51.12147771750289


Protein AMBP 8.806818181

818182 2

0.1325413151528118

7

0.005555555555555556

38.97398131913597

A0A0A0M Related Peroxiredoxin 16.95906432 3 0.4384498 0.03143274853 18.96367220

51

SI0 Proteins -1 (Fragment) 748538 8828766285

8011694 675963

Q6VFQ6 Single

Protein


71.42857142857143

4 20.544346900318832

6.7836734693878515

4.510410000496161

Appendix 3. All validated proteins in 7 days old blood sample.

Accession

Protein Inference

Class


Coverage (%)

#Validated Peptides

emPAI

NSAF

MW (kDa)


Complement C3

38.96572459410704

42 0.6726468435651758

0.011983136244567448

187.02987531966258

P01023 Single

Protein Alpha-2-


892809 40

1.0892961308540396

0.018982332037160137

163.18789146924135

P11277

Related and

Unrelated Proteins


erythrocytic

13.617220402433318

20 0.1867232263265372

6

0.0042177688439505485

246.3157334321971

A0A087WZE4

Related Proteins


erythrocytic 1

10.658436213991768

17 0.1284157410588553

4

0.0034657800100219667

280.9387841720546

C8C504 Single

Protein Beta-globin

95.23809523809523

23 33.14548873833603

1.0643221440986594

15.987342548192922

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

22 0.8410273525621605

0.052355876457586464

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

33.443708609271525

22 0.8410273525621605

0.040973426126460585

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

21 0.7906555296923772

0.20549480423022165

69.18136901532891


Apolipoprotein A-I

56.92883895131086

19 2.1622776601683795

0.1652404941943857

30.758932500112536

A0A0K0K1H8

Related and

Unrelated Proteins



33.0945558739255

19 0.7398280529303642

0.03377540862939089

77.02963611452142

P02787

Related and

Unrelated Proteins

Serotransferrin

32.23495702005731

19 0.7398280529303642

0.024019311068415247

77.01362996651181

P02787

Related and

Unrelated Proteins

Serotransferrin

32.664756446991404

19 0.7398280529303642

0.02076727854809005

77.01362996651181

P02730

Related and

Unrelated Proteins


21.514818880351264

13 0.5632550875412283

0.03809714782962014

101.72741218512044

Q3LR79 Single

Protein

Hemoglobin beta

(Fragment)

94.28571428571428

16 38.810717055349734

1.1922836562172698

11.474842789202496



92.51700680272108

17 12.593563908785255

0.4981073880703037

16.045289860997062

A0A0C4D Related Serum 30.62913907 19 0.6940088 0.03937058809 69.18136901

52

GB6 and Unrelated Proteins

albumin 2847682 022878714 203235 532891



159393 13

0.6907141034735806

0.03618560134024048

59.71875531355211

I1VZV6 Single

Protein Hemoglobin

alpha 1 92.95774647

887323 15

13.251026703029977

2.3448754749739096

15.269957551186833

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

40.909090909090914

18 1.2908676527677732

0.11001750049354113

45.11835747484806



44.806517311608964

14 0.7433288221999881

0.04154553627081096

55.89226078115152

P02671 Single


22.170900692840647

14 0.4364925262166947

7

0.020800481827672152

94.91441497589778

Q4TZM4 Single

Protein


90.0990099009901

14 24.118864315095795

1.2600347778541152

11.015638867792866

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

29.545454545454547

17 1.1877616239495525

0.09846991913892551

45.11835747484806

P16157 Single

Protein Ankyrin-1

6.273258904837853

8 0.1058999718195106

1

0.004302780918875907

206.1369244395263

Q9BWU5 Single

Protein


87.61904761904762

15 22.101297000831593

0.8998818548406703

11.493947863407726

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

39.39393939393939

16 1.0892961308540396

0.05707182473359479

45.11835747484806

A0A087WTM7

Related and

Unrelated Proteins


1.3812154696132597

4 0.0196629977857685

2

0.0014637904468412942

489.52739493234196

D9YZU8 Single

Protein Hemoglobin,

gamma A 89.11564625

850339 12

5.309573444801933

0.24460958668441302

16.11827504126487

A0A024R6I7

Related Proteins

Alpha-1-antitrypsin

40.19138755980861

13 1.1983926488622894

0.1048145667463854

46.678988138410034

A0A0G2JL54

Related Proteins

Complement C4-B

7.597173144876326

7 0.0982410543092073

8

0.0030211561064216264

187.59794049222188

P00918

Related and

Unrelated Proteins


54.61538461538461

11 1.5550970903525068

0.09428895946363199

29.227922186630536



36.659436008676785

11 0.7782794100389228

0.0442509226131786

52.30432646809949

A0A075B6N9

Related Proteins


(Fragment)

25.386313465783665

9 0.7252105499420405

0.03894164602257084

49.40858023156175


Peroxiredoxin-6

51.78571428571429

9 0.9952623149688795

0.050053146258503364

25.01919029056794

P06727 Single


n A-IV 17.17171717

171717 5

0.2426236719115049

0.01762295081967213

45.37146664308862

O95408 Single


70.17543859649122

7 55.23413251903491

2.700371221023367

6.26215476339843

A0A087WU08

Related Proteins


658363 9

0.9952623149688795

0.0317071552722898

31.3878684863898

53



e B chain

26.047904191616766

7 0.6065060038537282

0.017937068820461054

36.61514296895986


Purine nucleoside

phosphorylase

44.982698961937714

7 0.7782794100389228

0.019352446861097383

32.09715605245075

P60174 Single

Protein Triosephosphate isomerase

17.832167832167833

3 0.2589254117941673

0.01223776223776224

30.771696101193438

P32119

Related and

Unrelated Proteins

Peroxiredoxin-2

49.494949494949495

10 1.9935772947204895

0.16804455706894716

21.87823782783635

A0A075B6N8

Related and

Unrelated Proteins


(Fragment)

34.48275862068966

9 0.7508270317357244

0.02822759059122695

41.29932388898452

P02790 Single

Protein Hemopexin

28.78787878787879

8 0.5199110829529336

0.038049450549450525

51.64327297890149

Q6J1Z9 Single

Protein

Hemoglobin alpha 1

(Fragment) 90.0 9 9.0

1.0792442122953434

9.585181362001366

Q6MZU6

Related and

Unrelated Proteins



213

27.586206896551722

9 0.7012542798525889

0.04995489772862896

51.06619379122771



50.0 7 1.3357214690901222

0.09879796578825704

22.105426425215388

A0A087WV47

Related and

Unrelated Proteins


25.107296137339056

8 0.5671910803783076

0.10269080234833662

51.12147771750289

P01876

Related and

Unrelated Proteins


32.29461756373937

8 1.3101297000831598

0.019618994126092033

37.630638369583146

B7ZKJ8

Related and

Unrelated Proteins


807486 4

0.110336318167638

0.0052485004284490155

103.81622401008842

Q86YQ1 Single

Protein

Hemoglobin alpha-2

(Fragment)

87.91208791208791

6 4.623413251903491

0.6500600240096058

9.665182424630526



31.443298969072163

7 1.4484367468222272

0.13544067764846984

21.38873696621423

A0A087WT59

Related Proteins


783784 4

0.9306977288832501

0.12127396413110697

20.132907042394372

P02787

Related and

Unrelated Proteins

Serotransferrin

9.025787965616045

6 0.1911031332830068

8

0.024999999999999998

77.01362996651181

D6RBJ7

Related and

Unrelated Proteins


30.747126436781606

5 0.3433993325989002

0.013553639846743288

38.77189341127183



40.79601990049751

7 1.1544346900318838

0.05678845961864828

23.49675931597699

E9PFZ2 Related

and Ceruloplasmin

4.968287526427061

3 0.0746078283213174

0.0061950439648281366

108.75141618854433

54

Unrelated Proteins


Angiotensinogen

5.154639175257731

2 0.1406249238513208

6

0.016666666666666666

53.12051471946386



0.591715976331361

2 0.0093278530753431

18

8.104384471999352E-4

515.282910917923

Q86YQ4 Single

Protein

Alpha-1 globin

(Fragment)

90.9090909090909

5 3.2169650342858223

0.8393980340312006

9.476096235395074

Q9HAR8 Single

Protein

Mutant beta-globin

(Fragment)

96.7741935483871

4 20.544346900318832

2.4040342060153015

3.2907445311221695



60.16949152542372

6 3.6415888336127784

0.4572033898305086

13.248254100518784

A0A0G2JPD4

Related and

Unrelated Proteins


(Fragment)

20.489296636085626

6 0.637893706954064

0.034402332361516005

35.91786587959916

P02787

Related and

Unrelated Proteins

Serotransferrin

6.73352435530086

5 0.1568875283162822

8

0.030216802168021674

77.01362996651181

P19652 Single


glycoprotein 2 21.39303482

5870647 4

0.5199110829529336

0.05419222903885479

23.587631690019066


Delta-aminolevulini

c acid dehydratase

22.12121212121212

5 0.4174741629268053

0.02967948717948718

36.27141416930379

A0A0C4DGL8

Related Proteins


9683 2

0.1450475699382818

3

0.00606060606060606

38.42734787385203


Plasma protease C1

inhibitor

9.392265193370166

4 0.216483949025851

0.007893685051958433

59.454555992296385



10.424710424710424

2 0.1497569953977358

0.025096525096525095

29.986510368450627

B2M1S7 Single

Protein

Beta-globin Showa

Yakushiji variant

(Fragment)

100.0 4 20.544346900318832

1.6641534391534425

4.4933946847851916

A0A075B6H6

Related and

Unrelated Proteins


(Fragment)

79.43925233644859

5 1.8480358684358014

0.5171784601691172

11.711667188377447

A0A087WV45

Related Proteins


064749 4

1.0309176209047357

0.10558410414525522

15.110588167739953



21.467391304347828

4 0.3894954943731377

0.037849872773536894

39.386724601822

G3V3A0

Related and

Unrelated Proteins


psin

9.268292682926829

2 0.2451970847350328

8

0.026829268292682926

23.39110667577842


Kininogen-1 4.347826086

9565215 2

0.0690192046030009

3

0.012287334593572778

71.91215085210732


Adenylate kinase

24.742268041237114

3 0.2589254117941673

0.014175257731958761

21.621307247002466

55

isoenzyme 1

P08603

Related and

Unrelated Proteins

Complement factor H

2.518277822908205

2 0.0372250954070572

1

0.0029354207436399216

139.00469816836323

M0R009 Related Proteins

Alpha-1B-glycoprotein (Fragment)

10.130718954248366

2 0.1859710123376696

6

0.01507537688442211

33.43379931857088



29.381443298969074

4 0.6681005372000588

0.17965367965367965

21.38873696621423



15.979381443298967

4 0.6681005372000588

0.19182900432900435

21.38873696621423

A0A0B4J231

Related and

Unrelated Proteins


like polypeptide 5

32.093023255813954

4 0.6237767391887217

0.06905430711610487

23.135670373513683


Glyceraldehyde-3-


e

25.76923076923077

4 0.4251026703029981

0.05292792792792794

27.853060805444024



26.865671641791046

4 1.0309176209047357

0.12263242375601918

14.680972242798342

A0A0A0MRQ5

Related and

Unrelated Proteins

Peroxiredoxin-1

38.144329896907216

4 1.3101297000831598

0.039846179021436744

10.669507125347321


Antithrombin-III

7.543103448275862

2 0.0857111194022042

2

0.00646551724137931

52.56886431029427


Selenium-binding

protein 1

10.805084745762713

3 0.1620301363862613

0.0069209039548022615

52.35762502132521

P26447 Single


A4 18.81188118

811881 2

0.3593563908785255

6

0.034653465346534656

11.720732061372276

A0A087WTE1

Related Proteins

Inter-alpha-trypsin


H2

2.751322751322751

2 0.0481131341546856

55

0.006130790190735695

106.58970315928195


Beta-2-glycoprotein 1

19.420289855072465

4 0.3111339374215642

6

0.010338164251207732

38.27265933588615



8.393285371702639

2 0.0944997993195435

5

0.008393285371702638

44.58612676613191

A0A024R6I7

Related and

Unrelated Proteins

Alpha-1-antitrypsin

10.76555023923445

3 0.1993539462092342

0.05445075757575755

46.678988138410034


Apolipoprotein A-II

28.999999999999996

2 0.4677992676220694

7

0.2866666666666666

11.16789735900159

C9J5S7 Related Proteins


trans isomerase

26.446280991735538

3 0.7782794100389228

0.038016528925619845

13.199402292131417


Alpha-synuclein

31.30434782608696

2 0.3593563908785255

0.07391304347826087

11.769249433169955

56

6



873303 2

0.110336318167638

0.004456824512534818

49.07441753459848



97427 2

0.0784173969290393

7

0.00558659217877095

65.36674524764112

B7UCU6

Related and

Unrelated Proteins


19.047619047619047

2 0.5199110829529336

0.6580952380952414

11.578876214589096

A0A087WV47

Related and

Unrelated Proteins


6.866952789699571

2 0.1188722115874221

2

0.03757338551859099

51.12147771750289

A0A0B4J231

Related and

Unrelated Proteins


like polypeptide 5

13.023255813953488

2 0.2742749857031337

0.05280898876404492

23.135670373513683


Alpha-1-antitrypsin

5.980861244019139

2 0.1288378916846890

7

0.026515151515151516

46.707019438538325

A0A075B6P5

Related and

Unrelated Proteins

Protein IGKV2-28

(Fragment)

30.833333333333336

2 1.1544346900318838

0.10852713178294572

12.948457741318867

C9JTX5

Related and

Unrelated Proteins


(Fragment) 33.75 2

0.9306977288832501

0.046875 8.528166114

106645

P05109 Single


A8 23.65591397

8494624 2

0.3894954943731377

0.04838709677419355

10.827649221794548

E9PFZ2

Related and

Unrelated Proteins


536998 2

0.0491397291363098

2

0.002997601918465228

108.75141618854433

P00352

Related and

Unrelated Proteins

Retinal dehydrogenas

e 1

5.389221556886228

2 0.0890229622637301

9

0.005361930294906166

54.82694780360638


Complement C3

38.96572459410704

42 0.6726468435651758

0.011983136244567448

187.02987531966258

P01023 Single

Protein Alpha-2-


892809 40

1.0892961308540396

0.018982332037160137

163.18789146924135

P11277

Related and

Unrelated Proteins


erythrocytic

13.617220402433318

20 0.1867232263265372

6

0.0042177688439505485

246.3157334321971

A0A087WZE4

Related Proteins


erythrocytic 1

10.658436213991768

17 0.1284157410588553

4

0.0034657800100219667

280.9387841720546

C8C504 Single

Protein Beta-globin

95.23809523809523

23 33.14548873833603

1.0643221440986594

15.987342548192922

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

22 0.8410273525621605

0.052355876457586464

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

33.443708609271525

22 0.8410273525621605

0.040973426126460585

69.18136901532891

A0A0C4DGB6

Related and

Serum albumin

35.264900662251655

21 0.7906555296923772

0.20549480423022165

69.18136901532891

57

Unrelated Proteins

(A0A0C4DGB6_HUMAN)


Apolipoprotein A-I

56.92883895131086

19 2.1622776601683795

0.1652404941943857

30.758932500112536

A0A0K0K1H8

Related and

Unrelated Proteins



33.0945558739255

19 0.7398280529303642

0.03377540862939089

77.02963611452142

Appendix 4. All validated proteins in one day old saliva sample.

Accession

Protein Inference

Class


Coverage (%)

#Validated Peptides

emPAI

NSAF

MW (kDa)

A8K2U0 Unrelated Proteins

Alpha-2-macroglobulin-like protein 1

7.2214580467675376

7 0.1609534723271461

9

0.004905847373637266

161.00406439261246


Alpha-amylase 1

35.61643835616438

12 0.7378008287493754

0.011299818465682208

57.73091248903519


Alpha-amylase 1

26.614481409001954

10 0.5848931924611136

0.032871872666955196

57.73091248903519

H0YKL9 Related Proteins

Annexin (Fragment)

46.36363636363636

4 1.0309176209047357

0.09402597402597403

12.05628484306039

H0YL33 Related Proteins

Annexin (Fragment)

42.95774647887324

4 0.4924955450518296

0.04144869215291754

16.422326680576667


Annexin A1 35.54913294

797688 9

0.5264179671752336

0.09116088631984576

38.689979125461925


Annexin A1 22.54335260

115607 7

0.3894954943731377

0.06519749518304435

38.689979125461925


Annexin A1 8.381502890

17341 3

0.1513953993264474

0.012524084778420038

38.689979125461925


Annexin A2 34.80825958

702065 9

0.4896248995834391

3

0.01449155564199811

38.579814745475446

Q96DR5 Single

Protein

BPI fold-containing

family A member 2

24.899598393574298

5 0.7782794100389228

0.01979166666666667

26.99474719539754

Q8TDL5 Single

Protein

BPI fold-containing

family B member 1

33.057851239669425

11 1.06198600950222

0.0333488064813366

52.40847237175294

Q8N4F0 Single

Protein

BPI fold-containing

family B member 2

19.65065502183406

5 0.4677992676220694

7

0.05413832199546482

49.14172224203288

F8W148 Related Proteins


19.25133689839572

2 0.4677992676220694

7

0.04205607476635514

21.0494281284372


Cathepsin G 27.05882352

9411764 6

0.4839817889675653

0.11816993464052285

28.819069590686258

B4DQ50 Related Proteins

cDNA FLJ56823,

highly similar to Protein-glutamine gamma-

glutamyltransferase E (EC 2.3.2.13)

9.75609756097561

5 0.2648552168552962

0.01735749630486472

58.757343311769674

58

A8K2N8 Related Proteins

cDNA FLJ77935,

highly similar to Homo sapiens

guanylate binding protein family,

member 6 (GBP6), mRNA

7.424960505529226

3 0.0978437670592489

1

0.006497175141242938

72.31878032663606

A0A087WVQ9

Related and

Unrelated Proteins

Elongation factor 1-alpha

1

5.442176870748299

2 0.0841458689358347

6

0.004076086956521739

47.85292586470423


Galectin-3-binding protein

7.17948717948718

3 0.1620301363862613

0.003386004514672686

65.28928569959861

F8WE04

Related and

Unrelated Proteins

Heat shock protein beta-1

13.978494623655912

2 0.2216773489967920

3

0.045045045045045036

20.39351236294829

P01876

Related and

Unrelated Proteins


27.762039660056658

8 1.3101297000831598

0.0873453603579128

37.630638369583146

P01876

Related and

Unrelated Proteins


23.79603399433428

8 1.3101297000831598

0.07897715952109273

37.630638369583146

A0A0G2JMB2

Related and

Unrelated Proteins


(Fragment)

19.705882352941178

4 0.5505157798326246

0.05065502183406112

36.48504939159779

A0A087WV47

Related and

Unrelated Proteins


9.656652360515022

3 0.1835067295194687

8

0.01187214611872146

51.12147771750289

A0A075B6H6

Related and

Unrelated Proteins


(Fragment)

79.43925233644859

5 1.8480358684358014

0.2401869158878504

11.711667188377447

A1A4E9 Unrelated Proteins

Keratin 13 43.23144104

803493 25

2.4034148825183075

0.05969603479713928

49.55539934926395

O76011 Related Proteins

Keratin, type I cuticular Ha4

6.192660550458716

2 0.1158839925077483

9

0.009316770186335406

49.39222835129151



30.406852248394006

11 0.7343248017724648

0.05603970482487109

52.21397052719633

P13645 Single

Protein

Keratin, type I cytoskeletal

10

40.06849315068493

23 2.0857568527160524

0.04090458865775523

58.79169361540863



13

52.40174672489083

30 3.3480834671045114

0.14208560162064462

49.55737861381936

P02533 Single

Protein


14

58.26271186440678

30 2.5938136638046276

0.07079219228164929

51.52938407421822

P08779 Single

Protein


16

64.27061310782241

30 3.2169650342858223

0.0670095521349188

51.236202521320465

F5GWP8 Related Keratin, type I 26.36103151 10 0.6876124 0.01977262894 40.26237172

59

and Unrelated Proteins

cytoskeletal 17

8624642 757881477 1683384 672955

C9JM50

Related and

Unrelated Proteins


19 (Fragment)

9.693877551020408

2 0.2451970847350328

8

0.024092970521541936

20.83054632446172

P35527 Single

Protein Keratin, type I cytoskeletal 9

14.285714285714285

5 0.2589254117941673

0.011071987480438188

62.02681468242336

Q9NSB2 Related Proteins

Keratin, type II cuticular

Hb4

38.83333333333333

17 0.7782794100389228

0.02703092965937998

64.80146264176814


Keratin, type II cytoskeletal

1

32.142857142857146

26 1.5118864315095801

0.07067735734666779

65.99900087210398



1

31.987577639751557

24 1.3400803470013467

0.01855447460828886

65.99900087210398



16.588419405320813

11 0.476490881614259

0.009021635107839432

65.39321717101762

Q01546 Single

Protein


2 oral

21.786833855799376

15 0.7154378963428789

0.029619499569468553

65.80008965822827

P12035 Single

Protein


3

12.10191082802548

10 0.4773776525985111

7

0.012150872780268033

64.37756213683015

F8VX05 Related Proteins


4

53.84615384615384

6 2.511191734215131

0.09299219299219298

7.783852222922241



4

57.303370786516865

44 4.411695265464636

0.3026775414591496

57.249828579886504



4

7.116104868913857

6 0.2589254117941673

0.01755477939357032

57.249828579886504

F8VV57 Related Proteins

Keratin, type II cytoskeletal 5 (Fragment)

37.121212121212125

4 1.3101297000831598

0.0689922480620155

12.14979750828232

F8W0C6 Related Proteins


48.60335195530727

13 2.1622776601683795

0.03806504199904183

20.643608929250135



5

34.91525423728814

24 1.3101297000831598

0.010840742651444782

62.33997694347283

P02538

Related and

Unrelated Proteins


6A

47.16312056737589

32 2.0538555088334154

0.023024637601976136

60.00827426891604

P02538

Related and

Unrelated Proteins


6A

9.397163120567376

4 0.1497569953977358

0.016474654377880184

60.00827426891604

P04259

Related and

Unrelated Proteins


6B

17.5531914893617

13 0.5738730431619035

0.007594720056995347

60.03029098160933

P48668

Related and

Unrelated Proteins


6C

53.72340425531915

31 1.949150729647068

0.016751938655459726

59.98830556904431

H0YIC5 Related Keratin, type 14.73684210 6 0.4677992 0.02236525754 31.27068623

60

Proteins II cytoskeletal 73 (Fragment)

5263156 6762206947

0057658 2512684

Q8N1N4 Single

Protein


78

42.88461538461539

19 1.0733215734859551

0.01583255815452162

56.83049222411559

E7EQB2

Related and

Unrelated Proteins

Lactotransferrin (Fragment)

16.954022988505745

9 0.2875262659803554

7

0.005995712461229703

76.57688676417338

E7EQB2

Related and

Unrelated Proteins


11.350574712643677

6 0.1835067295194687

8

0.004732894819101716

76.57688676417338

E7EQB2

Related and

Unrelated Proteins


5.890804597701149

4 0.1188722115874221

2

0.002134646962233169

76.57688676417338

P30740

Related and

Unrelated Proteins


27.440633245382585

8 0.5505157798326246

0.0156984126984127

42.714702418590555


Myeloblastin 8.203125 2 0.3335214321633239

7

0.011986301369863013

27.789271098985488

P05164 Single

Protein Myeloperoxid

ase 9.932885906

040267 6

0.1721022975334802

8

0.004480796586059744

83.81483871714372

C3PTT6

Related and

Unrelated Proteins

Pancreatic adenocarcino

ma upregulated

factor

47.95918367346939

6 1.6826957952797255

0.06527777777777778

21.55300886308947

P01833 Single

Protein

Polymeric immunoglobul

in receptor

21.596858638743456

13 0.4405773606943357

3

0.011679751638768032

83.23165254290421

P05109 Single


A8 23.65591397

8494624 2

0.3894954943731377

0.07526881720430108

10.827649221794548

Q08188 Single

Protein

Protein-glutamine gamma-

glutamyltransferase E

28.282828282828284

15 0.7012542798525889

0.01309080795901598

76.58358425055039

P02787

Related and

Unrelated Proteins

Serotransferrin

13.753581661891118

7 0.2263306841775629

7

0.0021151116273067498

77.01362996651181

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

40.909090909090914

18 1.2908676527677732

0.025039988456087847

45.11835747484806

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

39.39393939393939

16 1.0892961308540396

0.022638280649507472

45.11835747484806

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

28.282828282828284

13 0.8197008586099834

0.020433838047474402

45.11835747484806

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

33.44370860927152

21 0.7906555296923772

0.024472496230653194

69.18136901532891

61

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

22 0.8410273525621605

0.013126754666477575

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

21 0.7906555296923772

0.031193520323226573

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

30.629139072847686

19 0.6940088022878714

0.014158728420040324

69.18136901532891

C9JDS9 Related Proteins


(Fragment)

23.717948717948715

2 0.4677992676220694

7

0.012962962962962963

17.65980705160292

Appendix 5. All validated proteins in one week old saliva sample.

Accession

Protein Inference

Class


Coverage (%)

#Validated Peptides

emPAI

NSAF

MW (kDa)

P60709

Related and

Unrelated Proteins


17.333333333333336

4 0.2915496650148839

0.012000000000000002

41.709730139029176

C9JTX5

Related and

Unrelated Proteins


(Fragment) 33.75 2

0.9306977288832501

0.015625 8.528166114

106645

I3L1U9 Related Proteins


(Fragment)

21.02803738317757

3 0.4125375446227544

0.017912772585669784

23.82995491331281

I3L1U9

Related and

Unrelated Proteins


(Fragment)

14.018691588785046

2 0.2589254117941673

0.053738317757009345

23.82995491331281

I3L4N8

Related and

Unrelated Proteins


(Fragment)

9.50413223140496

2 0.2451970847350328

8

0.008264462809917356

26.917436121169494

A0A024R6I7

Related Proteins

Alpha-1-antitrypsin

20.813397129186605

6 0.4384498882876628

5

0.013778409090909095

46.678988138410034

A8K2U0 Unrelated Proteins


8.872077028885833

8 0.1859710123376696

6

0.0028525378376716335

161.00406439261246

A8K2U0

Related and

Unrelated Proteins


6.121045392022008

5 0.1124904621939377

7

0.003014535844070036

161.00406439261246


Alpha-amylase 1

38.16046966731898

17 1.1877616239495525

0.018212733605126877

57.73091248903519


Alpha-amylase 1

29.9412915851272

15 0.9952623149688795

0.023635227502940503

57.73091248903519


Annexin (Fragment)

54.54545454545454

6 1.8942661247167512

0.05628492719401809

12.05628484306039


Annexin (Fragment)

45.07042253521127

8 1.2275429519995575

0.04700542024485685

16.422326680576667


Annexin (Fragment)

42.95774647887324

6 0.8233480008684411

0.055046948356807464

16.422326680576667

P04083 Related Annexin A1 40.46242774 12 0.7575106 0.04241729689 38.68997912

62

Proteins 566474 248547918 7065616 5461925


Annexin A1 22.54335260

115607 7

0.3894954943731377

0.047663776493256274

38.689979125461925


Annexin A1 10.98265895

9537572 5

0.2648552168552962

0.021290944123314063

38.689979125461925


Annexin A2 32.44837758

1120944 10

0.5570684047537309

0.010374816748621512

38.579814745475446


ATP synthase subunit alpha, mitochondrial

6.329113924050633

2 0.0722672220103233

2

0.007383966244725738

59.713593485620585

Q562R1 Single

Protein Beta-actin-like

protein 2 11.17021276

5957446 4

0.2826498305280605

0.007092198581560282

41.97596729003312

Q8TDL5 Single

Protein

BPI fold-containing

family B member 1

32.64462809917355

12 1.202201949987375

0.018371755419948198

52.40847237175294

Q8N4F0 Single

Protein

BPI fold-containing

family B member 2

21.397379912663755

6 0.5848931924611136

0.046712018140589576

49.14172224203288



411765 7

0.5848931924611136

0.024463118580765653

28.819069590686258

B3KSL2

Related and

Unrelated Proteins

cDNA FLJ36533 fis,

clone TRACH200442

8, highly similar to

Lactotransferrin (EC 3.4.21.-)

(Fragment)

27.83357245337159

16 0.5671910803783076

0.007601771062670419

76.70396132609598

B4E1U2 Related Proteins

cDNA FLJ53421,




26.490066225165563

9 0.318256738556407

0.004984721161191751

69.15420835722563

B0AZP3 Related Proteins

cDNA, FLJ79483,




19.589257503949447

7 0.2362470354499415

2

0.004180076044482824

72.28891128123597

P01024

Related and

Unrelated Proteins

Complement C3

9.320505111244739

9 0.1165349973889215

9

0.003083679590270928

187.02987531966258

F8WE04

Related and

Unrelated Proteins

Heat shock protein beta-1

18.27956989247312

3 0.3503140378698730

3

0.02195945945945946

20.39351236294829

P01876 Related

and Ig alpha-1

chain C region 16.71388101

983003 4

0.5199110829529336

0.010011954572624028

37.630638369583146

63

Unrelated Proteins

A0A075B6H6

Related and

Unrelated Proteins


(Fragment)

60.747663551401864

4 1.3101297000831598

0.026479750778816202

11.711667188377447


Junction plakoglobin

14.630872483221477

6 0.2409377607517195

8

0.005118694362017805

81.69273021332828

A1A4E9 Unrelated Proteins

Keratin 13 8.515283842

79476 6

0.3417128354799117

0.02486452671610999

49.55539934926395



8.653846153846153

3 0.1993539462092342

0.004275227877712349

47.20705069770084



36.402569593147746

16 1.2275429519995575

0.019729003815018072

52.21397052719633

P13645 Single

Protein


10

59.24657534246576

38 5.4344382633289925

0.06138780152088599

58.79169361540863

K7EMD9 Related Proteins


13 (Fragment)

23.636363636363633

5 0.7301957388458942

0.14370296370296348

19.20172933573258

P13646

Related and

Unrelated Proteins


13

58.29694323144105

36 4.833879397552119

0.07084789166092717

49.55737861381936

P02533 Single

Protein


14

66.52542372881356

40 4.50478980785497

0.040893038124746706

51.52938407421822

P08779 Single

Protein


16

66.38477801268499

34 4.108969774506928

0.08616791555915368

51.236202521320465

F5GWP8

Related and

Unrelated Proteins


17

32.378223495702

11 0.7782794100389228

0.0122549521647342

40.26237172672955

C9JM50

Related and

Unrelated Proteins


19 (Fragment)

9.693877551020408

2 0.2451970847350328

8

0.009566326530612245

20.83054632446172

P35527 Single


46.06741573033708

31 3.1686938347033538

0.032695992229512326

62.02681468242336



Hb4 49.0 22

1.1063445423241212

0.018366257401291743

64.80146264176814



1

44.409937888198755

44 3.752510411057904

0.07001459599988424

65.99900087210398



1

44.25465838509317

42 3.4274518434944925

0.019124388122095014

65.99900087210398

P35908 Single

Protein


52.895148669796555

34 2.334854751235186

0.01867898503002453

65.39321717101762

Q01546 Single

Protein


2 oral

25.54858934169279

18 0.9109529749704406

0.022339240977388604

65.80008965822827

P12035 Single

Protein


3

17.515923566878982

15 0.7957144943716399

0.017491630746450677

64.37756213683015

F8VX05 Related Proteins


4

53.84615384615384

8 4.3366992312063095

0.1666819291819291

7.783852222922241

64



4

57.67790262172285

50 5.812920690579613

0.11313900535763279

57.249828579886504



37.121212121212125

4 1.3101297000831598

0.05232558139534884

12.14979750828232



50.279329608938546

15 2.7750532053243937

0.03977384552889407

20.643608929250135

H0YI76 Related Proteins


49.75124378109453

15 2.5938136638046276

0.053792105802683364

23.09540181798727

H0YIN9 Related Proteins


17.857142857142858

5 0.5848931924611136

0.0076402905484538024

21.951672834646317

P02538

Related and

Unrelated Proteins


6A

47.695035460992905

38 2.7649358067924683

0.02982048393288893

60.00827426891604

P02538

Related and

Unrelated Proteins


6A

9.397163120567376

4 0.1497569953977358

0.01528417818740398

60.00827426891604

P04259

Related and

Unrelated Proteins


6B

17.5531914893617

16 0.7475284000076838

0.008287214379493335

60.03029098160933

P48668

Related and

Unrelated Proteins


6C

52.659574468085104

34 2.2745491628777286

0.016313025069223844

59.98830556904431

Q8N1N4 Single

Protein


78 30.0 13

0.6468978654828685

0.013024097049226038

56.83049222411559

C9JCF5

Related and

Unrelated Proteins


33.51648351648351

4 0.5199110829529336

0.031004709576138153

20.330700568898102

E7EQB2

Related and

Unrelated Proteins


54.310344827586206

31 1.3880794998622172

0.00921900328794767

76.57688676417338

E7EQB2

Related and

Unrelated Proteins


25.57471264367816

15 0.5237960002208242

0.014518519682884447

76.57688676417338



40.89709762532981

13 1.0395004690050569

0.030679745635627996

42.714702418590555

Q9HC84 Related Proteins

Mucin-5B 0.711558486

6365846 3

0.0251593260625877

5

0.0011342407259140647

595.9601181160408

P08246 Single

Protein Neutrophil

elastase 23.59550561

7977526 4

0.4924955450518296

0.010646108663729809

28.499788498823822

P05109 Single


A8 23.65591397

8494624 2

0.3894954943731377

0.04838709677419355

10.827649221794548

Q08188 Single

Protein



5.194805194805195

2 0.0734188827013531

0.005351681957186544

76.58358425055039

A0A087W Related Serum 22.22222222 6 0.3182567 0.00910006449 45.11835747

65

WT3 and Unrelated Proteins

albumin 222222 38556407 7791766 484806

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

8.080808080808081

2 0.0964781961431850

9

0.003787878787878788

45.11835747484806

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

18.70860927152318

7 0.2143317475937911

4

0.0038583588045508575

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

16.225165562913908

7 0.2143317475937911

4

0.008825246221769395

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

2.4834437086092715

2 0.0570520810009829

1

0.0028973509933774835

69.18136901532891



(Fragment)

32.69230769230769

3 0.7782794100389228

0.023478835978835978

17.65980705160292

A0A0A0MR02

Related Proteins

Voltage-dependent

anion-selective channel

protein 2 (Fragment)

14.539007092198581

3 0.2915496650148839

0.01590594744121715

30.328869561053356

Appendix 6. All validated proteins in three weeks old saliva sample.

Accession

Protein Inference

Class


Coverage (%)

#Validated Peptides

emPAI

NSAF

MW (kDa)

B0AZS6 Related Proteins

14-3-3 protein

zeta/delta

22.023809523809522

2 0.2915496650148839

0.026785714285714284

19.060395339266766

P60709

Related and

Unrelated Proteins


17.6 5 0.37685716485275833

0.006828282828282827

41.709730139029176

A0A024R6I7

Related Proteins

Alpha-1-antitrypsin

38.995215311004785

14 1.3357214690901222

0.034123676985892866

46.678988138410034

P01023 Single

Protein

Alpha-2-macroglobuli

n

5.563093622795115

5 0.09647819614318509

0.005975924333619948

163.18789146924135

A8K2U0 Single

Protein

Alpha-2-macroglobulin-like protein

1

20.97661623108666

23 0.6329126856447997

0.012726190530940045

161.00406439261246


Alpha-actinin-4

10.867178924259058

7 0.16939481814731705

0.004791916766706683

104.78847372042017


Alpha-amylase 1

45.79256360078278

18 1.2908676527677732

0.017815754826975377

57.73091248903519


Alpha-amylase 1

37.573385518590996

16 1.0892961308540396

0.037563825063825114

57.73091248903519

D6RA82 Related Proteins

Annexin 15.49295774

6478872 4

0.2589254117941673

0.013791079812206574

32.098530647992916


Annexin (Fragment)

54.54545454545454

5 1.4244620170823286

0.23088023088023074

12.05628484306039

66

H0YM50 Related Proteins

Annexin (Fragment)

42.74193548387097

14 1.5808615404180748

0.05414790023084957

28.35243878541001


Annexin A1 40.75144508

67052 13

0.8420699693267162

0.111610010092669

38.689979125461925


Annexin A1 29.19075144

5086704 10

0.5998587196060581

0.0944382849007126

38.689979125461925


Annexin A1 10.98265895

9537572 5

0.2648552168552962

0.04609826589595377

38.689979125461925


Annexin A2 36.87315634

218289 11

0.6275654480267538

0.05662621980945771

38.579814745475446


ATP synthase subunit beta, mitochondrial

10.01890359168242

4 0.20679264063932856

0.008451656524678837

56.524605849238945

Q562R1 Single

Protein Beta-actin-

like protein 2 11.17021276

5957448 3

0.20526093687084246

0.010638297872340422

41.97596729003312

Q96DR5 Single

Protein

BPI fold-containing

family A member 2

17.269076305220885

3 0.4125375446227544

0.02964743589743589

26.99474719539754

Q8TDL5 Single

Protein

BPI fold-containing

family B member 1

39.25619834710744

13 1.3519526350709588

0.017428669055175074

52.40847237175294

Q8N4F0 Single

Protein

BPI fold-containing

family B member 2

21.39737991266375

7 0.7113283041617808

0.05046971169420148

49.14172224203288

F8W148 Related Proteins


19.25133689839572

2 0.46779926762206947

0.056074766355140186

21.0494281284372

E9PQ63 Related Proteins

Carbonyl reductase [NADPH] 1

15.168539325842698

2 0.35935639087852556

0.01 19.00973458

34827



823529 9

0.8077686769634342

0.055639589169000914

28.819069590686258

B4DQ50 Related Proteins

cDNA FLJ56823,

highly similar to Protein-glutamine gamma-

glutamyltransferase E (EC

2.3.2.13)

6.378986866791745

3 0.1513953993264474

0.00755735492577598

58.757343311769674


Complement C3

8.057726999398678

8 0.10294331179161298

0.0035953427196365063

187.02987531966258

P12532 Single

Protein

Creatine kinase U-type, mitochondrial

8.393285371702639

2 0.08571111940220422

0.006527415143603133

47.00720625799705

Q9UGM3 Single

Protein

Deleted in malignant

brain tumors 1 protein

1.0360547036883547

2 0.03272814726171824

0.0040650406504065045

260.5688045493978

P15924 Single

Protein Desmoplakin

3.6224312086381047

6 0.03124778549427165

0.002487652090109626

331.5687042418961

Q16610 Single

Protein

Extracellular matrix

protein 1

10.37037037037037

3 0.14815362149688283

0.00629555997349238

60.635275149574795

F8WE04

Related and

Unrelated Proteins

Heat shock protein beta-

1

18.27956989247312

3 0.35031403786987303

0.052364864864864864

20.39351236294829

67


Histone H4 41.74757281

553398 4

0.46779926762206947

0.03802588996763755

11.360381729559025

P01876

Related and

Unrelated Proteins


21.246458923512748

6 0.8738174228603839

0.01536245600053131

37.630638369583146

A0A0G2JMB2

Related and

Unrelated Proteins


(Fragment)

19.705882352941178

4 0.5505157798326246

0.024599708879184848

36.48504939159779

A0A075B6H6

Related and

Unrelated Proteins


(Fragment)

79.43925233644859

5 1.8480358684358014

0.1459501557632398

11.711667188377447

D6RD17 Related Proteins

Immunoglobulin J chain

(Fragment)

13.924050632911392

2 0.2589254117941673

0.014227642276422764

18.110050897845266


Junction plakoglobin

25.503355704697984

14 0.6548170999431815

0.01536694018148024

81.69273021332828



55.03211991434689

21 1.8610255691043776

0.0609852152082874

52.21397052719633

P13645 Single

Protein


10

48.97260273972603

26 2.5743040182210506

0.061645886221465454

58.79169361540863

K7EMD9 Related Proteins


13 (Fragment)

18.787878787878785

4 0.5505157798326246

0.06070707070707076

19.20172933573258

P13646

Related and

Unrelated Proteins


13

40.61135371179039

20 1.6639858572587722

0.027271583980691398

49.55737861381936

K7ERE3

Related and

Unrelated Proteins


13

31.08433734939759

16 1.4040991835099716

0.02068739243662859

45.23223677712046

P02533 Single

Protein


14

55.50847457627119

24 1.7825594022071245

0.06805756935821794

51.52938407421822

P08779 Single

Protein


16

68.92177589852008

33 3.86967525

1658631 0.07589838127

230322 51.23620252

1320465

F5GWP8

Related and

Unrelated Proteins


17

7.736389684813753

2 0.11033631

8167638 0.00900532132

623823 40.26237172

672955

F5GWP8

Related and

Unrelated Proteins


17

8.022922636103152

3 0.16998910658906596

0.011246418338108884

40.26237172672955

F5GWP8

Related and

Unrelated Proteins


17

22.636103151862464

8 0.5199110829529336

0.01448122558314239

40.26237172672955



34.02889245585875

14 0.9054607179632472

0.02699504460067839

62.02681468242336



Hb4

63.83333333333333

33 2.0569907009353776

0.06387756583233463

64.80146264176814



1

36.33540372670807

33 2.21878751

1821236 0.09234496109

924112 65.99900087

210398

P04264 Related Keratin, type 36.18012422 31 1.99863134 0.03715559188 65.99900087

68

Proteins II cytoskeletal 1

360248 85755667 736077 210398

P35908 Single

Protein


32.70735524256651

22 1.1800253234900517

0.021890729594864256

65.39321717101762

Q01546 Single

Protein


2 oral

32.9153605015674

27 1.6416483203860923

0.04003354097912183

65.80008965822827

P12035 Single

Protein


3

14.649681528662422

12 0.5973122800602544

0.041079397861950924

64.37756213683015



4

51.49812734082397

31 2.28599324

7600655 0.14461868300

500147 57.24982857

9886504



37.121212121212125

4 1.3101297000831598

0.12286821705426357

12.14979750828232



48.60335195530726

12 1.8942661247167512

0.07975541948714984

20.643608929250135



49.75124378109453

14 2.3000347911252845

0.03732172646381498

23.09540181798727

P02538

Related and

Unrelated Proteins


6A

47.340425531914896

34 2.2745491628777286

0.03514860914390704

60.00827426891604

P02538

Related and

Unrelated Proteins


6A

9.397163120567376

4 0.1497569953977358

0.024014336917562724

60.00827426891604

P04259

Related and

Unrelated Proteins


6B

17.5531914893617

14 0.6297508346206442

0.010502703516885679

60.03029098160933

P48668

Related and

Unrelated Proteins


6C

53.723404255319146

34 2.2745491628777286

0.016279644616316077

59.98830556904431

Q8N1N4 Single

Protein


78

15.192307692307693

8 0.35935639087852556

0.012384434406173537

56.83049222411559

E7EQB2

Related and

Unrelated Proteins


25.28735632183908

14 0.4816025176256622

0.010914261146381048

76.57688676417338

E7EQB2

Related and

Unrelated Proteins


15.086206896551724

9 0.28752626598035547

0.015634960880904082

76.57688676417338

E7EQB2

Related and

Unrelated Proteins


10.057471264367816

6 0.18350672951946878

0.007262588943623421

76.57688676417338

E7EQB2

Related and

Unrelated Proteins


8.620689655172413

4 0.11887221158742212

0.005923458725182864

76.57688676417338



44.06332453825858

14 1.1544346900318838

0.04148739114915583

42.714702418590555

Q9HC84 Single Mucin-5B 1.614022908 6 0.05095164 0.00172137710 595.9601181

69

Protein 7122526 3813099246

16813453 160408


Myeloblastin 27.734375 4 0.7782794100389228

0.05165525114155255

27.789271098985488

P05164 Single

Protein Myeloperoxid

ase 4.832214765

100671 3

0.08263673387405435

0.005926979611190138

83.81483871714372

P08246 Single

Protein Neutrophil

elastase 35.58052434

456929 7

1.015337685941733

0.05328298720369201

28.499788498823822


Neutrophil gelatinase-associated lipocalin

12.121212121212121

2 0.22167734899679203

0.012269938650306749

22.57371613770579

C3PTT6

Related and

Unrelated Proteins

Pancreatic adenocarcino

ma upregulated

factor

47.95918367346939

6 1.6826957952797255

0.09078282828282824

21.55300886308947

P01833 Single

Protein

Polymeric immunoglobu

lin receptor

5.49738219895288

3 0.08789095479256037

0.004553734061930784

83.23165254290421

P05109 Single


A8 23.65591397

8494624 2

0.3894954943731377

0.12365591397849462

10.827649221794548


Protein S100-A9

26.31578947368421

2 0.4251026703029981

0.05263157894736842

13.233508230835282

Q08188 Single

Protein



14.141414141414142

7 0.2814223889440839

0.013810009222853255

76.58358425055039


Serpin B3 11.53846153

846154 4

0.216483949025851

0.013677324916518003

44.536544995847535

A0A024QZX5

Related Proteins

Serpin B6 9.210526315

789473 2

0.11887221158742212

0.005263157894736842

42.99625360584759

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

34.343434343434346

12 0.7378008287493754

0.01192559335721838

45.11835747484806

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

34.84848484848485

12 0.7378008287493754

0.010662967094592107

45.11835747484806

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

28.14569536423841

15 0.5160826983319609

0.013175929060987523

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

27.81456953642384

14 0.47460159321419093

0.03359869768349508

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

28.14569536423841

13 0.4342554407501811

0.027316182997497662

69.18136901532891

P68363

Related and

Unrelated Proteins

Tubulin alpha-1B chain

9.090909090909092

3 0.19377664171443643

0.00975975975975976

50.119604615111854



(Fragment)

19.230769230769234

2 0.46779926762206947

0.0308641975308642

17.65980705160292

A0A075B736

Related and

Tubulin beta-8 chain

3.43980343980344

2 0.1497569953977358

0.0035211267605633804

45.66683080042066

70

Unrelated Proteins

A0A0A0MR02

Related Proteins

Voltage-dependent



15.24822695035461

3 0.2915496650148839

0.027662517289073305

30.328869561053356

Q9Y277 Single

Protein

Voltage-dependent


protein 3

10.60070671378092

2 0.1659144011798317

0.008264462809917354

30.639285645312086

P25311 Single

Protein Zinc-alpha-2-glycoprotein

21.140939597315437

4 0.32194114846602906

0.010889774236387784

34.23710469367686

Appendix 7. All validated proteins in fresh semen sample.

Accession

Protein Inference

Class


Coverage (%)

#Validated Peptides

emPAI

NSAF

MW (kDa)

P10323 Single

Protein Acrosin

8.788598574821853

2 0.1497569953977358

0.004424778761061947

45.817403494908454

A6NL76

Related and

Unrelated Proteins

Actin, alpha skeletal muscle

4.724409448818897

2 0.2022644346174129

5

0.008858267716535433

28.147013604276893

F5H7P4

Related and

Unrelated Proteins

A-kinase anchor


19.1358024691358

3 0.4677992676220694

7

0.011316872427983538

18.2717306218222

Q5JQC9 Related Proteins

A-kinase anchor

protein 4

31.850117096018735

23 0.6559515234819178

0.010743683417059502

94.4165133446506

A0A024R6I7

Related Proteins

Alpha-1-antitrypsin

27.51196172248804

9 0.7252105499420405

0.011496663059163059

46.678988138410034

P03973 Single

Protein Antileukoprot

einase 20.45454545

4545457 2

0.2742749857031337

0.011363636363636364

14.315908944800055

A1YZ08 Related Proteins

Beta-globin (Fragment)

16.19047619047619

2 0.6681005372000588

0.02222222222222222

11.411847146306945

J3KNB4 Related Proteins

Cathelicidin antimicrobial

peptide

29.47976878612717

5 0.5085907086001784

0.025144508670520236

19.578268250841486

E5RG36 Related Proteins


14.634146341463413

3 0.7782794100389228

0.027777777777777776

9.318919755181668



42.142857142857146

5 0.7782794100389228

0.045782312925170064

16.166165820637204

H0YC35 Related Proteins


13.028169014084508

3 0.2689610031679221

5

0.021985815602836876

33.50827632671542



17.6056338028169

3 0.2689610031679221

5

0.009361702127659573

33.50827632671542

P12277

Related and

Unrelated Proteins

Creatine kinase B-type

6.036745406824147

2 0.1253355826007649

4

0.00487012987012987

42.617318186065454

P54107 Related Cysteine-rich 23.69477911 5 0.7301957 0.02294520547 28.46233643

71

Proteins secretory protein 1

6465863 388458942 9452058 95561

P81605 Single

Protein Dermcidin 20.0 2

0.4251026703029981

0.05714285714285714

11.27682755014452

E7EMS2

Related and

Unrelated Proteins

Epididymal secretory protein E1

25.37313432835821

5 0.7782794100389228

0.039271708683473384

22.426332974690606

E7EMS2

Related and

Unrelated Proteins


28.35820895522388

5 0.7782794100389228

0.039271708683473384

22.426332974690606


Epididymal secretory

protein E3-beta

31.292517006802722

3 0.4677992676220694

7

0.015873015873015876

17.572542210621645

Q16610 Single

Protein

Extracellular matrix protein

1

42.96296296296296

17 1.1877616239495525

0.011345410946334811

60.635275149574795


Fibronectin 3.855825649

6228 7

0.0947774484233141

5

0.001420512820512821

262.4601018836126



7912825 3

0.0395704655308148

9

9.435897435897435E-4

262.4601018836126

K7EJY8 Related Proteins


(Fragment)

15.972222222222221

2 0.5848931924611136

0.0273972602739726

15.600550849698703



20.68376068376068

8 0.4924955450518296

0.012289046544125555

65.28928569959861

P20142 Single

Protein Gastricsin

5.154639175257731

2 0.3593563908785255

6

0.04054054054054054

42.39860601325266

A0A0A0MTN3

Related Proteins

Glutathione S-transferase

Mu 3

19.81132075471698

4 0.4454397707459274

4

0.010422282120395327

24.898433659591365



Mu 3

15.11111111111111

3 0.2915496650148839

0.009185185185185183

26.542137718578985


Glyceraldehyde-3-


e

10.76923076923077

2 0.1937766417144364

3

0.006756756756756757

27.853060805444024


Glyceraldehyde-3-


e, testis-specific

11.519607843137255

3 0.1787686347935873

0.00853413654618474

44.47278994794948

G3V1N2

Related and

Unrelated Proteins

HCG1745306, isoform CRA_a

8.181818181818182

2 0.6681005372000588

0.045454545454545456

11.940248402513872


Heat shock-related 70 kDa

protein 2

4.694835680751173

2 0.0570520810009829

1

0.0024671052631578946

69.97786578851715

F8W6P5 Related

and Unrelated

Hemoglobin subunit beta (Fragment)

24.444444444444443

2 0.6681005372000588

0.08148148148148147

9.664011956204076

72

Proteins


Histone H2AX 22.37762237

7622377 2

0.2022644346174129

5

0.027972027972027972

15.13541509128964

Q96A08 Single

Protein Histone H2B

type 1-A 27.55905511

8110237 5

0.53174046370208

0.017716535433070862

14.158740271128844


Histone H4 29.12621359

2233007 3

0.3335214321633239

7

0.022653721682847898

11.360381729559025

P01876

Related and

Unrelated Proteins


5.382436260623229

2 0.2328467394420661

3

0.006276150627615063

37.630638369583146

A0A0G2JN06

Related and

Unrelated Proteins


(Fragment)

7.975460122699387

2 0.1787686347935873

0.004347826086956522

35.87775615263485

A0A075B6H6

Related and

Unrelated Proteins


(Fragment)

47.66355140186916

3 0.8738174228603839

0.040498442367601244

11.711667188377447

C9J4N6 Related Proteins

Isocitrate dehydrogenas

e [NADP] cytoplasmic (Fragment)

14.012738853503185

2 0.2328467394420661

3

0.008492569002123142

17.708279879975482



e [NADP] cytoplasmic

14.492753623188406

5 0.2532543355087804

0.005744125326370757

46.62951442889963

A0A0B4J1X3

Related and

Unrelated Proteins

Kallikrein 3, (Prostate specific

antigen), isoform CRA_f

46.666666666666664

3 1.6826957952797255

0.052222222222222225

9.939295244151824

P29622

Related and

Unrelated Proteins

Kallistatin 4.683840749

414521 2

0.1188722115874221

2

0.003978779840848806

48.51115813677777



40.38461538461539

17 1.8013567611988681

0.016917374451138015

47.20705069770084


Keratin, type I cuticular Ha3-I

4.702970297029703

3 0.1835067295194687

8

0.0023765432098765433

45.91139143371252


Keratin, type I cuticular Ha3-

II

31.683168316831683

16 1.5719138090593452

0.012657806754216351

46.18453981219836



21.55963302752294

13 1.0395004690050569

0.01149717324066475

49.39222835129151



4.496788008565311

4 0.2216773489967920

3

0.0019430697180057791

52.21397052719633



5.79064587973274

2 0.1220184543019633

4

0.00469283276450512

49.71517718694274

P13645 Single

Protein


10

59.93150684931506

29 3.1401995764592234

0.047906600693310125

58.79169361540863

P02533 Single

Protein


14

34.74576271186441

17 1.0644996558525563

0.022440496169737353

51.52938407421822

P08779 Single Keratin, type I 34.67230443 19 1.4879023 0.02172154965 51.23620252

73

Protein cytoskeletal 16

97463 672388363 5470452 1320465

K7EPJ9 Related Proteins


17 (Fragment)

13.852813852813853

3 0.3335214321633239

7

0.014896867838044309

24.850220348241024

P35527 Single


28.08988764044944

12 0.7378008287493754

0.047004586441206156

62.02681468242336



Hb5

37.08086785009862

25 1.392147081462638

0.010629797754568116

55.766472822378276

O43790

Related and

Unrelated Proteins


Hb6

25.720164609053498

18 0.9952623149688795

0.009028859142731487

53.46629878272636



1

46.58385093167702

39 2.9810717055349722

0.0386707662314013

65.99900087210398



1

46.42857142857142

37 2.7087774117744745

0.01770754838608496

65.99900087210398

P35908 Single

Protein


38.96713615023474

23 1.2586355207213766

0.014882862439378827

65.39321717101762



11.1731843575419

2 0.1937766417144364

3

0.02304964539007092

20.643608929250135



10.945273631840797

2 0.1859710123376696

6

0.009271822704658523

23.09540181798727



25.37313432835821

5 0.53174046370208

0.013092656376238469

23.09540181798727

P02538

Related and

Unrelated Proteins


6A

23.936170212765955

14 0.6297508346206442

0.005963741688562697

60.00827426891604

P04259 Single

Protein


6B

37.765957446808514

23 1.2309243744089828

0.008459523616154664

60.03029098160933

O95678

Related and

Unrelated Proteins


75

5.9891107078039925

3 0.110336318167638

0.004112006137322593

59.52403085076316

P0C5Y4 Single

Protein

Keratin-associated protein 1-4

18.181818181818183

2 9.0 1.25 12.31459956

1097713

A0A087WY89

Related Proteins


23.62204724409449

2 99.0 0.20512820512

820515 13.23078217

2692193

Q9BYR4 Single

Protein


20.51282051282051

2 1.5118864315095801

0.020588235294117647

20.487390980336997

C9JCF5

Related and

Unrelated Proteins


38.46153846153846

6 0.8738174228603839

0.028268777925371327

20.330700568898102

E7EQB2

Related and

Unrelated Proteins


59.339080459770116

38 1.9067965371321285

0.013106851572035795

76.57688676417338

E7EQB2 Related

and Lactotransferrin (Fragment)

28.16091954022988

17 0.6118219775228428

0.008434298498947139

76.57688676417338

74

Unrelated Proteins

E7EQB2

Related and

Unrelated Proteins


24.71264367816092

16 0.5671910803783076

0.010177119543455395

76.57688676417338

E7EQB2

Related and

Unrelated Proteins


14.942528735632186

8 0.2518750258625291

0.00974821906363488

76.57688676417338

F5H245 Related Proteins


e

18.553459119496853

5 0.3894954943731377

0.008194514554938583

34.78345530992753

F5GZ11 Related Proteins


e C chain

25.748502994011975

4 0.6681005372000588

0.0124750499001996

18.273454494679733

A0A0G2JN42

Related Proteins

Mucin-6 4.100041000

410004 7

0.1504548126912206

3

0.004102170231202491

256.87106254481546

C9JR96 Related Proteins

Neprilysin (Fragment)

7.725321888412018

2 0.1787686347935873

0.003787878787878788

26.206501468672535


Peptidyl-prolyl cis-trans

isomerase B

19.90740740740741

4 0.3219411484660290

6

0.014274691358024692

23.72753554152188

G3V264 Related Proteins

Plasma serine protease inhibitor

(Fragment)

15.11627906976744

3 0.5399265260594921

0.025362318840579712

19.00183930625142

P05154

Related and

Unrelated Proteins


19.704433497536947

8 0.5347813444464111

0.012889781159011926

45.6456892396854

P05154

Related and

Unrelated Proteins


5.665024630541872

2 0.1130421932675225

2

0.009861932938856018

45.6456892396854

P13796

Related and

Unrelated Proteins

Plastin-2 2.870813397

1291865 2

0.0670112884883351

5

0.0012626262626262627

70.24389151037927

P12273 Single

Protein

Prolactin-inducible protein

65.75342465753425

9 2.651741272548377

0.12902261361165474

16.561795793109898

C9JIZ6

Related and

Unrelated Proteins

Prosaposin 18.02656546

4895636 8

0.4924955450518296

0.005188124632569078

58.402024132169025

C9JIZ6 Related Proteins

Prosaposin 10.62618595

825427 5

0.2843862635897427

0.004774968972499836

58.402024132169025

A0A0J9YXB8

Related and

Unrelated Proteins

Prosaposin (Fragment)

22.15568862275449

4 0.6237767391887217

0.011477045908183632

18.84223226985116

M0QZF9 Related Proteins

Prostate-specific antigen

(Fragment)

28.488372093023255

4 1.0309176209047357

0.07951070336391435

19.077636924772218



(Fragment)

25.0 2 0.4251026703029981

0.011467889908256881

19.077636924772218

P15309 Single Prostatic acid 21.76165803 10 0.8632463 0.02812852981 44.53747290

75

Protein phosphatase 1088082 119315603 392307 479376

P33764 Single


A3 34.65346534

653465 3

0.9952623149688795

0.03300330033003301

11.705427107785646

P49221

Related and

Unrelated Proteins


glutamyltransferase 4

28.50877192982456

13 0.5738730431619035

0.005911085256414506

77.09605137624143

P49221

Related and

Unrelated Proteins



25.146198830409354

12 0.5199110829529336

0.005976360669002827

77.09605137624143

A6NCN2 Single

Protein

Putative keratin-87

protein

43.92156862745098

16 1.5719138090593452

0.02454025852217254

29.098648422651973

P04279 Single

Protein Semenogelin-

1 46.96969696

969697 21

1.209338675153079

0.02094908501664457

52.099881419104314

Q02383

Related and

Unrelated Proteins

Semenogelin-2

47.93814432989691

23 1.0073891800546302

0.02278506623973967

65.40541810235962

Q02383

Related and

Unrelated Proteins

Semenogelin-2

25.601374570446733

14 0.5283067326587692

0.023977254131893274

65.40541810235962



15.671641791044777

2 0.4251026703029981

0.016853932584269662

14.680972242798342

P02787

Related and

Unrelated Proteins

Serotransferrin

9.169054441260744

6 0.1911031332830068

8

0.004521228545618789

77.01362996651181

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

39.39393939393939

16 1.0892961308540396

0.031570906409462265

45.11835747484806

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

40.909090909090914

16 1.0892961308540396

0.014730420537636993

45.11835747484806

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

29.545454545454547

12 0.7378008287493754

0.06611060095908577

45.11835747484806

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

24 0.946061794205562

0.021300016170924474

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

33.443708609271525

24 0.946061794205562

0.013899067497554938

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

23 0.8928161540172448

0.009830790547386548

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

30.629139072847682

21 0.7906555296923772

0.019144742655375656

69.18136901532891


Small proline-rich protein

43.05555555555556

2 0.9306977288832501

0.023148148148148147

7.959674810476288

76

2A



facilitated glucose


5.846774193548387

2 0.1450475699382818

3

0.0030737704918032786

53.88925360580755


Sulfhydryl oxidase 1

7.228915662650602

4 0.1188722115874221

2

0.0029467232437529463

82.525505695686



8.041958041958042

2 0.1659144011798317

0.005244755244755245

30.771696101193438


Tubulin alpha-3C/D chain

17.77777777777778

6 0.4251026703029981

0.0106425702811245

49.92760276463705

P04350

Related and

Unrelated Proteins

Tubulin beta-4A chain

5.63063063063063

2 0.1406249238513208

6

0.006230529595015576

49.5539008224435

P25311 Single


29.86577181208054

8 0.7475284000076838

0.02192657939669893

34.23710469367686

Appendix 8. All validated proteins in one week old semen sample.

Accession

Protein Inference

Class


Coverage (%)

#Validated Peptides

emPAI

NSAF

MW (kDa)

B0AZS6 Related Proteins

14-3-3 protein zeta/delta

18.452380952380953

2 0.2915496650148839

0.008928571428571428

19.228432227324365

E5RGE1 Related Proteins

14-3-3 protein zeta/delta (Fragment)

44.230769230769226

3 1.6826957952797255

0.052884615384615384

5.900867956717122

A6NL76

Related and

Unrelated Proteins

Actin, alpha skeletal muscle

4.330708661417323

2 0.2022644346174129

5

0.005905511811023622

28.343056640344084

I3L1U9

Related and

Unrelated Proteins


(Fragment)

3.7383177570093453

2 0.2589254117941673

0.004672897196261682

23.941979505351206

I3L1U9

Related and

Unrelated Proteins


(Fragment)

14.018691588785046

2 0.2589254117941673

0.005841121495327103

23.941979505351206

O75969

Related and

Unrelated Proteins

A-kinase anchor

protein 3

3.9859320046893316

3 0.0715193052376064

1

0.005015959872321022

95.79756464191061


A-kinase anchor

protein 4

31.733021077283368

24 0.692666615037876

0.01645890406275211

95.71879922709695

G3V3A0

Related and

Unrelated Proteins


psin

9.268292682926829

2 0.2451970847350328

8

0.012195121951219513

23.587149711845623

A0A024R6I7

Related Proteins

Alpha-1-antitrypsin

45.45454545454545

14 1.3357214690901222

0.012158375084511445

47.029064988530074


Alpha-1-antitrypsin

5.980861244019139

2 0.1288378916846890

7

0.0037878787878787876

47.05709628865835

P06733 Related Alpha-enolase 6.221198156 2 0.0890229 0.00345622119 47.50339920

77


682028 6226373019

81566822 969828

D6RBL5 Related Proteins

Annexin 6.538461538

461539 2

0.1601553017399717

2

0.005128205128205128

29.557028662930108

E9PHT9 Related Proteins

Annexin 14.72392638

0368098 2

0.2451970847350328

8

0.008179959100204498

18.35249589254833

H0YKS4 Related Proteins

Annexin (Fragment)

15.340909090909092

2 0.2328467394420661

3

0.008522727272727272

19.72811294758306

A1YZ08 Related Proteins

Beta-globin (Fragment)

16.19047619047619

2 0.6681005372000588

0.030952380952380953

11.481862516330946

C9JE88 Related Proteins

Carboxypeptidase E

(Fragment)

17.687074829931973

2 0.3894954943731377

0.008503401360544218

16.655435195112936



peptide

35.83815028901734

6 0.637893706954064

0.04672447013487476

19.732302064894288

C9JH19 Related Proteins

Cathepsin D (Fragment)

7.023411371237458

2 0.2022644346174129

5

0.006818181818181818

32.94940452458041

E7ERK6 Related Proteins


10.294117647058822

2 0.2022644346174129

5

0.03592814371257485

23.89271049633231



42.142857142857146

5 0.7782794100389228

0.08129251700680269

16.348205782699605



17.6056338028169

3 0.2689610031679221

5

0.0198581560283688

33.858353176835415


Cystatin-C 36.98630136

9863014 4

0.8478497974222909

0.024543378995433792

15.915108539527141


Cysteine-rich secretory protein 1

34.53815261044177

8 1.4040991835099716

0.059776581865622944

28.7283948456473

P81605 Single

Protein Dermcidin 20.0 2

0.4251026703029981

0.1357142857142857

11.374849068178118

P27487 Single

Protein Dipeptidyl

peptidase 4 3.655352480

417754 3

0.1052951411260216

7

0.005817335660267597

89.11868106265338

E7EMS2

Related and

Unrelated Proteins


25.37313432835821

5 0.7782794100389228

0.05460317460317456

22.706394454786608

E7EMS2

Related and

Unrelated Proteins


28.35820895522388

5 0.7782794100389228

0.04872082166199813

22.706394454786608


Epididymal secretory

protein E3-beta

12.925170068027212

2 0.2915496650148839

0.017006802721088437

17.796591394698446

A0A087WY45

Related Proteins

Epididymal sperm-binding

protein 1

21.363636363636363

4 0.7190722018585745

0.01648841354723708

25.912657899809762

Q16610 Single

Protein

Extracellular matrix protein

1

10.37037037037037

5 0.2589254117941673

0.012160371106693173

61.09737659173322



362114 16

0.2299505200127773

8

0.003948484848484847

265.13468901853

78



5264038 8

0.1090313431155935

9

0.003977289377289378

265.13468901853

H3BQN4 Related Proteins

Fructose-bisphosphate

aldolase

4.43213296398892

2 0.1188722115874221

2

0.004155124653739612

39.581448596571214



15.213675213675213

7 0.4196258964747483

5

0.009185209072342257

66.04545169585785

A0A087WT12

Related Proteins

Glutathione peroxidase

8.583690987124463

2 0.1029433117916129

8

0.006437768240343348

27.07075946024807

A0A0A0MTN3

Related Proteins


Mu 3

34.43396226415094

6 0.7378008287493754

0.010413093196112066

25.038464399639373



Mu 3

22.22222222222222

4 0.4065272421052370

5

0.00726984126984127

26.696171532631798


Glyceraldehyde-3-


e

20.384615384615383

3 0.3043213867190053

0.008258258258258258

28.11911921153523


Glyceraldehyde-3-


e, testis-specific

20.588235294117645

6 0.3894954943731377

0.01641566265060241

44.83686987207431

A0A087WU08

Related Proteins


661921 2

0.1659144011798317

0.006072874493927126

31.653926892481003

G3V1N2

Related and

Unrelated Proteins


8.181818181818182

2 0.6681005372000588

0.11363636363636363

12.08027914256187

P07900

Related and

Unrelated Proteins

Heat shock protein HSP

90-alpha

8.19672131147541

5 0.1124904621939377

7

0.003784421116689487

85.19481044264995

F8W6P5

Related and

Unrelated Proteins


24.444444444444443

2 0.6681005372000588

0.06666666666666668

9.734027326228073

F8W6P5

Related and

Unrelated Proteins


21.11111111111111

2 0.6681005372000588

0.03333333333333333

9.734027326228073

P68871

Related and

Unrelated Proteins

Hemoglobin subunit beta

14.965986394557824

2 0.3894954943731377

0.008503401360544218

16.05830554829089

E9PEW8

Related and

Unrelated Proteins

Hemoglobin subunit delta (Fragment)

16.346153846153847

2 0.5848931924611136

0.019230769230769232

11.422787674368216



9.523809523809524

2 0.3593563908785255

6

0.015306122448979591

16.129308305025862


Histone H2AX 22.37762237

7622377 2

0.2022644346174129

5

0.038461538461538464

15.233436609323244

Q96A08 Single Histone H2B 35.43307086 7 0.8165997 0.04628796400 14.41079560

79

Protein type 1-A 614173 883753275 4499436 3215245


Histone H4 50.48543689

320388 5

0.6155980984398741

0.044336569579288033

11.388387877568626

A0A0G2JN06

Related and

Unrelated Proteins


(Fragment)

11.65644171779141

3 0.2798022139979537

0.007971014492753625

36.40987296481729

A0A0G2JN06

Related and

Unrelated Proteins


(Fragment)

7.975460122699387

2 0.1787686347935873

0.01594202898550725

36.40987296481729

A0A075B6N8

Related and

Unrelated Proteins


(Fragment)

5.570291777188329

2 0.1325413151528118

7

0.003272727272727272

41.873449923181354

A0A075B6H6

Related and

Unrelated Proteins


(Fragment)

79.43925233644859

5 1.8480358684358014

0.048909657320872275

11.935716372454246

A0A075B6L0

Related and

Unrelated Proteins

Ig lambda-3 chain C regions

(Fragment)

28.30188679245283

2 0.5848931924611136

0.012578616352201257

11.535570877437527

A0A0B4J231

Related and

Unrelated Proteins

Immunoglobulin lambda-like polypeptide 5

13.023255813953488

2 0.2742749857031337

0.007490636704119849

23.485747223633687



e [NADP] cytoplasmic

5.797101449275362

2 0.0944997993195435

5

0.0039164490861618795

46.937582057005244

P13645 Single

Protein


10

47.26027397260274

24 2.2406960357871677

0.04110284571876937

60.02396412783115

P02533 Single

Protein


14

19.06779661016949

9 0.4677992676220694

7

0.013770903330440724

52.25754392246785

P08779 Single

Protein


16

26.004228329809727

11 0.6949881513903466

0.013477315849512467

52.09039003561333

P35527 Single


46.3884430176565

24 2.019951720402016

0.04271799445788091

63.24508212084106



1

43.32298136645963

41 3.27335754210932

0.04726986085696493

67.25927753253609



1

43.7888198757764

41 3.27335754210932

0.045027708390597214

67.25927753253609



1

45.03105590062112

41 3.27335754210932

0.02709048865965556

67.25927753253609



31.29890453834116

19 0.9602329826462779

0.02388981589445987

66.56947538742092



10.614525139664805

2 0.1937766417144364

3

0.00911854103343465

20.797642743302937



29.850746268656714

6 0.6681005372000588

0.013638158414277821

23.22142948403047

P02538 Related

and Keratin, type II cytoskeletal

12.943262411347517

7 0.2766169490574077

0.008008076153237445

60.8484587092041

80

Unrelated Proteins

6A 5

H0YIC5 Related Proteins


5.964912280701754

2 0.1364636663857248

2

0.01809954751131222

31.676775378651893

O95678

Related and

Unrelated Proteins


75

5.9891107078039925

3 0.110336318167638

0.004163150492264417

60.47623988308964

P49223 Single

Protein

Kunitz-type protease

inhibitor 3

10.112359550561797

2 0.5848931924611136

0.03932584269662921

10.300968613824361

E7ER44

Related and

Unrelated Proteins

Lactotransferrin

22.88135593220339

16 0.5505157798326246

0.005016411911613034

78.61991452957133

C9JCF5

Related and

Unrelated Proteins


37.91208791208791

5 0.6876124757881477

0.03177152517811858

20.4147190129269

E7EQB2

Related and

Unrelated Proteins


58.90804597701149

39 1.9895770856517885

0.02092331723906281

77.27704046441345

E7EQB2

Related and

Unrelated Proteins


25.718390804597703

17 0.6118219775228428

0.013826185775666008

77.27704046441345

E7EQB2

Related and

Unrelated Proteins


24.71264367816092

16 0.5671910803783076

0.01129316686614199

77.27704046441345

E7EQB2

Related and

Unrelated Proteins


14.942528735632186

8 0.2518750258625291

0.011376777943715057

77.27704046441345



e 24.0 4

0.4924955450518296

0.008941798941798942

24.856767372954035



e

14.465408805031446

4 0.3010252169108314

0.009098939929328623

35.16153830805714

F5GZ11 Related Proteins


e C chain

20.35928143712575

3 0.4677992676220694

7

0.022027373823781008

18.469497530746935

A0A0G2JN42

Related Proteins

Mucin-6 8.446084460

844608 18

0.4339094903163154

0.01029776627682042

261.3940554483679


Neutrophil defensin 1

20.212765957446805

3 1.1544346900318838

0.12433862433862433

10.236187843494735

Q5T4C6

Related and

Unrelated Proteins

Outer dense fiber protein 2

(Fragment)

10.731707317073171

2 0.1188722115874221

2

0.01707317073170732

23.519176908481832


Peptidyl-prolyl cis-trans

isomerase B

39.351851851851855

8 0.7475284000076838

0.01466600529100529

23.853563207565088

A0A0A0MRQ5

Related and

Unrelated Proteins

Peroxiredoxin-1

19.587628865979383

2 0.5199110829529336

0.015463917525773196

10.767528643380924



6.474820143884892

2 0.0944997993195435

0.0035971223021582736

44.92220054224713

81

5



4.796163069544365

2 0.0926008611173783

1

0.008393285371702638

45.08940048266703



(Fragment)

15.11627906976744

2 0.3335214321633239

7

0.010869565217391304

19.26789771234262

P05154

Related and

Unrelated Proteins


17.733990147783253

7 0.4547578108480476

7

0.027574668920822755

46.17780605186783

P05154

Related and

Unrelated Proteins


5.665024630541872

2 0.1130421932675225

2

0.00690335305719921

46.17780605186783

P13796

Related and

Unrelated Proteins

Plastin-2 15.15151515

1515152 7

0.2548492322523297

0.004365079365079364

70.6919898785329

B4DV12

Related and

Unrelated Proteins

Polyubiquitin-B

23.52941176470588

2 0.2328467394420661

3

0.004357298474945533

17.288250487698054

P12273 Single

Protein


67.8082191780822

9 2.651741272548377

0.13393944335725155

16.617808089129095



(Fragment)

45.93023255813954

5 1.4244620170823286

0.07479248580166008

19.245673812829825

M0R1F0 Related Proteins


(Fragment)

39.69465648854962

7 1.0805675382171707

0.03593736593736593

28.937531405477543

P15309 Single

Protein Prostatic acid phosphatase

34.715025906735754

13 1.2456979955397744

0.03839685075640131

44.9575651249378

P05109 Single


A8 23.65591397

8494624 2

0.3894954943731377

0.016129032258064516

10.883661517813751

P49221

Related and

Unrelated Proteins



27.192982456140353

14 0.6297508346206442

0.007661527070861417

77.82421122449104

P49221

Related and

Unrelated Proteins



27.631578947368425

14 0.6297508346206442

0.0045987399345673915

77.82421122449104


Ras-related protein Rab-

3B

8.67579908675799

2 0.2115276586285883

6

0.005707762557077625

24.98016280066708


Semenogelin-1

9.090909090909092

3 0.119902793272475

0.0059781488352916926

52.870050489368396

Q02383

Related and

Unrelated Proteins

Semenogelin-2

18.04123711340206

8 0.2742749857031337

0.01161225658648339

66.48365480072933

P02787

Related and

Unrelated Proteins

Serotransferrin

11.031518624641834

8 0.2626001098748562

7

0.008519339738851928

77.58775600070864

82

P02787

Related and

Unrelated Proteins

Serotransferrin

4.584527220630372

4 0.1236548001387509

6

0.005555555555555556

77.58775600070864

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

39.39393939393939

19 1.3988329190194904

0.023499754725918646

45.34240665892489

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

40.909090909090914

18 1.2908676527677732

0.02053831534976497

45.34240665892489

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

29.545454545454547

17 1.1877616239495525

0.08613473346650724

45.34240665892489

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

23 0.8928161540172448

0.019978244272235022

69.55945201345854

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

33.443708609271525

23 0.8928161540172448

0.013898044857081628

69.55945201345854

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

22 0.8410273525621605

0.007684772421369718

69.55945201345854

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

30.629139072847682

20 0.7416619158620705

0.017509927256889657

69.55945201345854

Q9HBV2 Single

Protein

Sperm acrosome

membrane-associated protein 1

8.503401360544217

2 0.2328467394420661

3

0.011904761904761904

32.45897068733914



15.034965034965033

3 0.2589254117941673

0.00815850815850816

30.981742211265434


Tubulin alpha-3C/D chain

20.22222222222222

7 0.5117750706156623

0.008813826735513482

50.20766424473307

Q14508 Single

Protein

WAP four-disulfide core

domain protein 2

25.806451612903224

3 1.371373705661655

0.014784946236559139

13.109904292093264

P25311 Single


41.946308724832214

10 1.0092330025650473

0.02529089989249352

34.50316309976807

Appendix 9. All validated proteins in two weeks old semen sample.

Accession

Protein Inference

Class


Coverage (%)

#Validated Peptides

emPAI

NSAF

MW (kDa)


A-kinase anchor

protein 4

13.348946135831381

10 0.24519708473503288

0.00573272699185108

94.4165133446506

A0A024R6I7

Related Proteins

Alpha-1-antitrypsin

23.205741626794257

8 0.6237767391887217

0.009970238095238095

46.678988138410034

P15144 Related

and Unrelated

Aminopeptidase N

2.5853154084798344

2 0.05925372517728888

5

0.002074688796680498

109.47088086489971

83

Proteins


Beta globin (Fragment)

56.666666666666664

2 9.0 0.30555555555

555547 3.105616870

99756

C8C504 Single

Protein Beta-globin

95.23809523809523

17 12.593563908785255

0.09113337337605304

15.987342548192922



31.443298969072163

6 1.1544346900318838

0.039015151515151524

21.38873696621423



15.979381443298967

4 0.6681005372000588

0.044101731601731614

21.38873696621423



17.010309278350515

3 0.46779926762206947

0.026623376623376625

21.38873696621423



53.38983050847458

4 1.7825594022071245

0.0713276836158192

13.248254100518784



028463 2

0.08414586893583476

0.003092783505154639

59.71875531355211



peptide

31.213872832369944

5 0.5085907086001784

0.04007707129094412

19.578268250841486



42.142857142857146

5 0.7782794100389228

0.04180272108843538

16.166165820637204



17.6056338028169

3 0.26896100316792215

0.013049645390070926

33.50827632671542


Cysteine-rich secretory protein 1

34.53815261044177

6 0.9306977288832501

0.025913242009132424

28.4623364395561

P81605 Single

Protein Dermcidin

22.727272727272727

3 0.7012542798525889

0.16904761904761906

11.27682755014452

E7EMS2

Related and

Unrelated Proteins


25.37313432835821

5 0.7782794100389228

0.04492997198879551

22.426332974690606

E7EMS2

Related and

Unrelated Proteins


28.35820895522388

5 0.7782794100389228

0.04492997198879551

22.426332974690606



205365 11

0.15291622965094254

0.0023473748473748475

262.4601018836126

H0Y7Z1

Related and

Unrelated Proteins

Fibronectin (Fragment)

5.802357207615594

4 0.11735910194850852

0.0021684223495348977

120.69924410608232



9.914529914529915

4 0.22167734899679203

0.010346124905944318

65.28928569959861

A0A0A0MTN3

Related Proteins


Mu 3

9.433962264150944

2 0.20226443461741295

0.007075471698113208

24.898433659591365


Glyceraldehyde-3-


e, testis-specific

6.127450980392156

2 0.11588399250774839

0.004518072289156626

44.47278994794948

G3V1N2

Related and

Unrelated Proteins


27.27272727272727

4 1.7825594022071245

0.07575757575757575

11.940248402513872

G3V1N2 Related HCG1745306, 8.181818181 2 0.66810053 0.29545454545 11.94024840

84


isoform CRA_a

818182 72000588 454547 2513872

P07900

Related and

Unrelated Proteins

Heat shock protein HSP

90-alpha

3.415300546448088

2 0.04356262977539105

0.0021299254526091584

84.60668133444835

Q4TZM4

Related and

Unrelated Proteins


18.81188118811881

2 0.5848931924611136

0.03630363036303629

11.015638867792866



92.51700680272108

15 9.0 0.08311330198

635113 16.04528986

0997062


Histone H4 38.83495145

631068 4

0.46779926762206947

0.023462783171521034

11.360381729559025

A0A0G2JN06

Related and

Unrelated Proteins


(Fragment)

7.975460122699387

2 0.1787686347935873

0.007608695652173913

35.87775615263485

A0A075B6N8

Related and

Unrelated Proteins


(Fragment)

6.896551724137931

2 0.13254131515281187

0.008484848484848484

41.29932388898452

A0A075B6H6

Related and

Unrelated Proteins


(Fragment)

49.532710280373834

3 0.8738174228603839

0.02959501557632399

11.711667188377447

A0A0B4J1X3

Related and

Unrelated Proteins

Kallikrein 3, (Prostate specific

antigen), isoform CRA_f

46.666666666666664

3 1.6826957952797255

0.06555555555555556

9.939295244151824

P13645 Single

Protein


10

45.37671232876712

22 1.9382253839710089

0.03855687187377902

58.79169361540863

P02533 Single

Protein


14

15.889830508474576

8 0.40652724210523705

0.014081559621608156

51.52938407421822

P08779 Single

Protein


16

19.45031712473573

9 0.5399265260594921

0.014800206568499246

51.236202521320465



38.04173354735153

21 1.6302679918953817

0.06886960919822949

62.02681468242336



Hb4 8.0 4

0.14504756993828183

0.0036613942589338016

64.80146264176814



1

42.39130434782609

42 3.4274518434944925

0.04218357074981934

65.99900087210398



1

42.85714285714286

42 3.4274518434944925

0.046667875682554795

65.99900087210398



1

44.099378881987576

42 3.4274518434944925

0.019762046086142234

65.99900087210398

P35908 Single

Protein


45.22691705790297

24 1.3400803470013467

0.017632989006432412

65.39321717101762



24.378109452736318

5 0.53174046

370208 0.01677327647

4769016 23.09540181

798727

P02538 Related Keratin, type 13.82978723 8 0.32194114 0.00964113105 60.00827426

85


II cytoskeletal 6A

4042552 846602906 9537131 891604



6B

14.361702127659573

8 0.32194114846602906

0.007883559093236512

60.03029098160933

P49223 Single

Protein

Kunitz-type protease

inhibitor 3

24.719101123595504

4 1.5118864315095801

0.040262172284644196

10.244956317805164

E7EQB2

Related and

Unrelated Proteins


56.75287356321839

35 1.6719557914574237

0.018069572959375415

76.57688676417338

E7EQB2

Related and

Unrelated Proteins


24.71264367816092

16 0.5671910803783076

0.010202868370256859

76.57688676417338

E7EQB2

Related and

Unrelated Proteins


14.942528735632186

8 0.2518750258625291

0.0077747695941888335

76.57688676417338

E7EQB2

Related and

Unrelated Proteins


9.913793103448276

5 0.15073576110506148

0.006490603904397009

76.57688676417338



e 24.0 4

0.4924955450518296

0.008518518518518519

24.576705892858026



e

6.918238993710692

2 0.14062492385132086

0.00530035335689046

34.78345530992753



e A chain

6.626506024096386

2 0.12533558260076494

0.005611672278338945

36.66536097963206

A0A0G2JN42

Related Proteins

Mucin-6 11.56211562

1156211 20

0.4924955450518296

0.009394621720425095

256.87106254481546


Neutrophil defensin 1

20.212765957446805

3 1.1544346900318838

0.06878306878306878

10.194178621480336

P32119

Related and

Unrelated Proteins

Peroxiredoxin-2

23.232323232323232

4 0.5505157798326246

0.026219512195121957

21.87823782783635



(Fragment)

15.11627906976744

2 0.33352143216332397

0.014492753623188406

19.00183930625142

P05154

Related and

Unrelated Proteins


19.458128078817733

7 0.45475781084804767

0.01819878839109609

45.6456892396854

P05154

Related and

Unrelated Proteins


5.665024630541872

2 0.11304219326752252

0.010848126232741619

45.6456892396854

P13796

Related and

Unrelated Proteins

Plastin-2 2.870813397

1291865 2

0.06701128848833515

0.0025252525252525255

70.24389151037927

B4DV12

Related and

Unrelated Proteins

Polyubiquitin-B

23.52941176470588

2 0.23284673944206613

0.004901960784313725

17.204232043669254

86

P12273 Single

Protein


67.8082191780822

9 2.65174127

2548377 0.11627799521

635135 16.56179579

3109898



(Fragment)

28.488372093023255

4 1.0309176209047357

0.08103975535168192

19.077636924772218



(Fragment)

25.0 2 0.4251026703029981

0.022935779816513763

19.077636924772218

P15309 Single

Protein Prostatic acid phosphatase

19.4300518134715

7 0.5459277364194774

0.03256643481362582

44.53747290479376

P05109 Single


A8 23.65591397

8494624 2

0.3894954943731377

0.021505376344086023

10.827649221794548

P49221

Related and

Unrelated Proteins



10.818713450292398

6 0.23284673944206613

0.006176620724859624

77.09605137624143

Q02383

Related and

Unrelated Proteins

Semenogelin-2

8.075601374570446

4 0.12883789168468907

0.006300114547537228

65.40541810235962

P02787

Related and

Unrelated Proteins

Serotransferrin

12.034383954154727

8 0.26260010987485627

0.004137953284294748

77.01362996651181

P02787

Related and

Unrelated Proteins

Serotransferrin

5.014326647564469

3 0.0913767146512734

0.0014905149051490515

77.01362996651181

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

40.909090909090914

18 1.2908676527677732

0.020301118239581258

45.11835747484806

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

39.39393939393939

16 1.0892961308540396

0.024466815542912388

45.11835747484806

A0A087WWT3

Related and

Unrelated Proteins

Serum albumin

29.545454545454547

14 0.9054607179632472

0.08490954892470043

45.11835747484806

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

23 0.8928161540172448

0.01785082171675831

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

33.443708609271525

23 0.8928161540172448

0.018516638484375028

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

35.264900662251655

22 0.8410273525621605

0.007941904415063799

69.18136901532891

A0A0C4DGB6

Related and

Unrelated Proteins

Serum albumin

30.629139072847682

20 0.7416619158620705

0.014051571625427894

69.18136901532891

Age determination of body fluids by mass spectrometry - … · Age determination of body fluids by...

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