Proteomics between the principles

and applications

By

Aaser Abdelazim, PhD Assistant professor of Biochemistry

Zagazig University aaserabdelazim@yahoo.com

2018

What proteomics means?

100,000

proteins 20235 genes

The term “proteomics” was firstly

coined from merging “protein” and

“genomics” in the1990s

DNA (Gene)

RNA

PROTEIN

METABOLITE

Transcription

Translation

Enzymatic

reaction

Genomics/proteomics

Genomics

Metabolomics

Proteomics

Transcriptomics

Data bases statistics

21262

42108

Metagenomic studies Complete sequencing projects

Genome analysis projects

Non-metagenomic studies

ORGANISMS According to Genomes OnLine Database (https://gold.jgi.doe.gov/)

0

1000

2000

3000

4000

5000

6000

7000

16

5585

6343

5544 5559

5129

4679 4390

4043 3761

3520

3086

2417

1665

1040

519 219

59 22 1

# o

f p

rote

om

ics s

tud

ies

Year

Proteomics studies statistics

According to pubmed.gov /2016

Proteomics search statistics 29/10/2015

1

• Gene Expression and gene sequence

2

• Protein sequence data base

3

• Bioinformatics tools and their applications

4

• Microarray era and its applications

Stages of new Biology

What is the difference between proteomics and protein

chemistry?

Differences between protein chemistry and

proteomics

Protein chemistry Proteomics

Individual proteins Complex mixtures of proteins

Complete sequence analysis Partial sequence analysis

Emphasis on structural and

functions

Emphasis on identification by

database matching

Structural biology Systems biology

1

• The level of mRNA not necessarily predicts the level of

corresponding protein.

2

• Once formed, protein differ in stability and go to turn over.

3

• mRNA inform us nothing about the regularity status of

corresponding protein.

If we can measure gene expression why we use

proteomics ?

What is the challenge in proteomic techniques ?

１ • Proteins can not be amplified as genes.

2

• Cannot be hybridized with amino acids .

• Gene can be detected by oligonucleotides sequence.

3

• One gene can gives more than one protein.

• That due to post transitional modifications which varies from status to anther in the cell.

Proteomics in terms

1 2 3 4 5 6

Proteomics strategies

Bottom-up

Single Protein

Peptides

Top-down

Proteins

Proteins

Middle-down

Large peptides

Shotgun proteomics

Target

Depend on

Useful to study proteins modifications

Targets for shotgun proteomics

Data base

(data of all protein expressed )

Mass spectrometry

(protein mass, peptide mass and protein

sequence )

Soft ware

(matches mass data with specific protein

sequence )

Analytical protein separation

(allow investigation to target specific protein for

analysis )

1

2

3

4

Tools of proteomics

What is the fact which proteomics based on?

Peptidome

sample

Tissue

Fluids

Protein

extraction

Filtration

Organic solvents

Protein

separation

Chromatographic

separation

Electrophoresis

Protein

detection

ESI-MS/MS

MALDI-TOF

Data mining

SEQUEST

Mascot

Principles of proteomics steps

Separation Protein mixture Individual Protein

Separation

Digestion Digestion

MS analysis

MS data search

Peptides mixture Peptides

PROTEIN IDENTIFICATION

General flow of proteomics techniques

To answer this , imagine you copy a book on one paper , what is the

results?!

So we tend to increase the accuracy of analysis by decrease the complexity of proteins in mixture

Protein separation

Why we separate proteins rather used as mixture ?

1 D-SDS-PAGE

Reduction /denaturation

SDS-detergent binding

Apply to gel

2D SDS-PAGE

Load the strip

Focusing

Wash, add SDS

reluctant, join to

PAG

PAGE

Protein digestion

1

• Great molecular mass of proteins increase the error in mass measurement of most recent mass instruments.

2 • Not all masses of proteins can be measured.

3

• The sensitivity of measuring masses of intact proteins is less than that of peptides.

Why we don’t make MS simply on proteins ?

Why we digest proteins ?

Protease Cleavage specificity Common proteomic usage

Trypsin -K,R-↑-Z- not -K,R-↑-P- General protein digestion.

Endoproteinase Lys-C -K-↑-Z- Alternative to trypsin for increased

peptide length; multiple protease

digestion; 18O labeling.

Chymotrypsin -W,F,Y-↑-Z- and -L,M,A,D,E-↑-Z- at a slower

rate

Multiple protease digestion

Subtilisin broad specificity to native and denatured

proteins

Multiple protease digestion

Elastase -B-↑-Z- Multiple protease digestion

Endoproteinase Lys-N -Z-↑-K- Increase peptide length; create

higher charge state for ETD.

Endoproteinase Glu-C -E-↑-Z- and 3000 times slower at -D-↑-Z- Multiple protease digestion; 18O

labeling

Endoproteinase Arg-C -R-↑-Z- Multiple protease digestion

Endoproteinase Asp-N -Z-↑-D- and -Z-↑-cysteic acid- but not -Z-↑-C- Multiple protease digestion

Proteinase K -X-↑-Y- Nonspecific digestion of membrane-

bound proteins

Omp T -K,R-↑-K,R- Increased peptide length for middle-

down proteomics

B_ uncharged, non aromatic amino acid----- X_aliphatic, aromatic, hydrophobic amino acid---------------Z_ any amino acid

What we use to digest proteins ?

Success of digestion Staining techniques , acetic acid retard the penetration of trypsin into

gel and increase protein fixation in gel Residual components as SDS and acrylamide inhibit Trypsin .

Advantages

Display good activity in solution or in gel Mass instruments and lab nowadays were familiar to deal with tryptic peptides

Action

Cleavage the peptide bond formed by arginine or lysine (basic) followed by proline at C – terminal

Modifications

With TCPK (Tosylphenylalanylchloromethane) This to inhibits the residual chymotrypsin

Sources

Procaine pancreas Bovine pancreas

Trypsin digestion

In-gel trypsin digestion

• SDS remains one of the best protein solubilizers • It is detrimental to LC-MS peptide sensitivity. • it is a strong chaotropic agent.

(1) Organic solvents

• Commercially available surfactant as ProteaseMAX, Invitrosol, Rapigest, PPS Silent Surfactant.

• Volatile surfactant as Perfluorooctanoic acid and 1-butyl-3-methyl imidazolium tetrafluoroborate.

(2) MS-compatible surfactants

•Selectively cleaves proteins at aspartic acid residues. •Creating complementary peptides of similar length to trypsin digestion.

(3) Microwave heating of proteins under acidic

conditions

• Raising the pressure of proteolytic digestion has also improved the efficiency of proteolytic digestion. (4) Raising the pressure

Improvement of protein digestion

Protein identification

Proteins were identified by use high advanced mass spectrometers

MALDI – TOF

• Matrix assisted laser desorption ionization – time of flight

ESI- Tandem MS

• Elctrospray ionization

Types of mass instruments

The three main components of mass spectrometers

MALDI-TOF

1. Sample/matrix admixture placed on

plate or slide.

2. Laser then fires a beam of light at

target.

3. High energy from laser transferred to

peptides or proteins enable them to

ejected from source to gas phase.

4. Positive ions are usually formed by

accepting of proton when they ejected

from the matrix and these ions are

always of interest.

1. TOF simply measure the time that ions

will take to strike the detector.

2. This depends on ions m/z as greater

m/z the faster they fly.

3. Flying of ions in the TOF tube by this

way lead to poor resolution.

1. Reflectron focuses ions of the similar

m/z and allow them to reach detector

in the same time .

2. This improve the resolution of TOF.

Pros and cons of MALDI

In general there is no PERFECT MS instrument for analytical proteomics but MALDI-TOF deserves very high marks in four important categories.

It is very easy

Compatible with robotic sample preparation this increase the speed and the reproducibility of analyses.

High accuracy and resolution

High sensitivity it can acts on low quantities of peptides (femtomole)

/attomole (10-18 mole)

1 2 3 4

Cons of MALDI

ESI-Tandem MS source

1. Sample in aqueous

solution from HPLC.

2. All peptides

/proteins in acidic

medium (PH < 3.5)

will carry (+)

charges.

3. ESI is always done

in acidic medium .

Usually stainless steel needle

Fine mist of droplets contains

ions and HPLC mobile phase

(H2O, acetonitrile, acetic acid)

Separated peptide ions

Heated capillary Curtain of nitrogen gas

Triple quadrupole

Ion trap

quadrupole –time

of flight (Q-TOF)

ESI-MS analysis of bovine apomyoglobin. The “multicharge envelope” of signals from differently charged

forms of the protein.

ESI –MS spectrum Proteins in general may be single or multiple protonated For example 1. Protein with 20 kDa may accepts 10-30

protons in a solution.

2. If it have 20 protons its m/z will be 20,020/20 =

1001

3. If it have 19 protons its m/z will be 20,019/19 =

1053 and so on.

4. So ESI spectra will appears as so-called

(multicharge envelope) in which all the different

charge states of protein in solution are

represented .

ESI-MS analysis of bovine apomyoglobin. The deconvoluted spectrum indicating a single signal.

Charge –deconvolution algorthorism and software

can convert spectrum to one that represents the

actual protein mass. 16,949.0

ESI –MS spectrum

* amu= atomic mass unit = 1/12 mass of 12C ---------- one amu = 1.66x10-24 gram

4 metal rods magnetic filed

allows ions to flow between rods

According to the voltage, ions with

specific m/z will pass and others not.

Q1, Full scan:

yields signals

for all ions

(Q1): Q1 acts

as a mass

filter.

(q2) Ions

collide in q2

with argon

gas and go

fragmentation.

(Q3) Ions with

specific m/z were

analyzed according

to their m/z by Q3.

Triple quadrupole

Ion trap

Sequential ejection of ions based on their

m/z values.

Here full scan of all ions is occurred.

Collision with helium gas atoms induce the

fragmentation of ions

MS analysis is described here as (rocks in a can)

Automated data acquisition/MS-MS analysis in ESI

points Triple quadrupole MS

analyzer

Ion trap MS analyzer

Analysis technique On the fly On trapped ions

Collision gas Argon gas Helium gas

Ions flow strategy continuously according

to the voltage used.

Sequential according to

their m/z.

Fragmentation Less complete Much complete

Precursor ion signal Prominent Not seen

Mass resolution High Very high

Triple quadrupole Vs Ion trap analyzers

Other Mass Analyzers: Q-TOF MS Instruments.

Q3 in Quadrupole replaced by TOF

Higher mass resolution

Library

building

Virtual

digestion

Database of

sequences

(i.e. SwissProt)

Protein spot

from gel

Trypsin

digestion

MS analysis

and spectrum

generation

Matching

Lib

rary

Peptide identification by peptide mass

fingerprinting

Peptide identification by peptide mass

fingerprinting

This depends on

How we can do Peptide mass fingerprinting?!

to imagine this :

Trypsin Digestion

Total proteome

Peptides of specific

1. Length

2. Sequence

3. Most important Mass

Measured

peptide

mass by MS

Compared to

Matching of peptide mass with peptide mass list generated by virtual digestion

We will do this for all measured masses of tryptic peptides, one by one -------------gives indication about the unknown protein.

Although this is idealized example ….the success of PMF depends on : 1. Accurate measurement of peptide masses.

2. Accurate data base of protein sequence.

Success of peptide mass finger printing depend upon

Accurate measure of peptide mass

(1) Skill to do SDS- PAGE

(2) Skill of trypsin digestion

(3) High accuracy mass instrument

(4) High accuracy HPLC

Accurate data base

Good and strong soft ware To

provide accurate protein sequence

Success of peptide mass finger printing

Peptide mass matches for human Hb-α

Tryptic digestion of human hemoglobin alpha chain yields 14 tryptic peptides, of which the

peptide [VGAHAGEYGAEALER] has an exact monoisotopic mass of 1528.7348 Da.

Thus, the singly charged ion of this peptide has an m/z value of 1529.7348.

The results of searching this peptide against all mouse and human proteins in the SWISS-

PROT data base are illustrated in Table.

# hits= # peptide matches

When we use more stringent mass tolerance the results will be two proteins identified.

BUT THE QUESTION IS How we can Identify the right protein from these very similar matches?!

The answer: is the increase the number of peptides in the search (use multiple peptide matches)

Instead we search with ONE peptide we can search with TWO or THREE peptides

•The peptides in the sample are very huge to be calculated by human mind so always we

need soft ware for help.

•Several data base engines were used for identification e.g. SWISSPROT/ OWL/ NCBInr.

Soft ware for peptide mass finger printing

Data feeding

Peptide sequence analysis

Let us study the tandem mass analysis of the peptide AVAGCAGAR

Peptide bond

(C) (N)

Residue 72+99+ 71+57+103+71+57+71+168 =769

Average mass of each amino acid

1) Many bonds in the peptide subjected to collision by neutral gas but the most significant cleavages are along

peptide backbone .

2) Each peptide cleaved into y ions (in which + charge retained on the C terminus ) and b ions (in which + charge

retained on N terminus).

3) Other many cleavages are also appear but they are unusual as they need high much energy (like a, z, c and x).

Peptide ion fragmentation:

Peptide cleavage

Mass can detect this by making several cleavages of b and y series when you subtract

the mass of two successive series it will give you the mass of amino acid in between for

example the gap between y7 and y6 will give you 71 amu which is mass of Alanine and

so on

Bioinformatics tools in proteomics

BIOINFORMATICS Many programs and software are used to analyze the data obtained by MS

http://integratedproteomics.com/

ProteoIQ (http://www.bioinquire.com/)

Scaffold (http://www.proteomesoftware.com/)

MaxQuant (http://maxquant.org/)

Transproteomics Pipeline (http://tools.proteomecenter.org)

Proteome-Discoverer (www.thermoscientific.com)

pFind Studio (http://pfind.ict.ac.cn/)

Applications of proteomics

Applications of

proteomics

Protein expression

profiling Protein net

work mapping Mapping of

protein modifications

Protein mining Means simply identification of all proteins in sample

Identification of proteins in sample in particular sate of organism or cell as (disease, drug, exposure to chemicals or physical agents….etc)

Determine how proteins interacts with each other in living systems. Such as signal transduction cascades, complex biosynthetics , degradation pass ways.

Determine how and where protein were modified post transitional modification govern the targeting structure , function and turnover.

1- Protein mining (means discovering of proteins)

1. No cell in any organism contains all proteins encoded by its genes all together at one time.

2. In many tissues of higher organisms different genes and proteins are expressed as a common(which perform

vital functions) however others are expressed specially in close relation to the tissue e.g. contractile proteins

expressed in muscles, photoreceptors are express proteins as a result of light stimulation

3. However many proteomes are changed (posttranstional modification)or may be present extra cellular (CSF,

blood, urine..) as a results of many diseases.

4. Detection of these proteins can be used as a marker for diagnosis listed in the text

MS detections

Body parts

Protein markers Mining

Tissue

Abnormal proteins

detections Extra

expression

Fluids

Protein detection

2- Protein expression profiling

1. Biochemical studies revealed that the proteome of the cell is

continually changed, this changing is essential for life.

2. Other changes are induced by chemicals, environment, drugs, growth

and disease process.

3. The changes are used to study complicated pathologies like cancer .

4. So Protein profiling task is to measure the proteome in two or more

samples then compare them.

A B

C

Advantages of Protein profiling

Collection of cell for proteome analysis

1. Each type of selected cell should be representative for the proteome which we hope to study (kidney cells

are many but we should select one cell type )Many techniques are used to collect different cell types e.g

laser-captureMicrodissection which enable collection of unique type of cells

2. The problems of proeome analysis during collection of cells include

A. Proteome change due to stress condtions induced during cell manipulation which may produce

ROS which lead to protein oxidation.

B. Activation of internal proteases during cell hemogenization which lead to fragmentation of protein

C. Prepration,fixation and preservation manner of tissue samples e.g formaldehyde can prevent

protein digestion.

Mining approach

Use of proteomics as a tool for detection of new biomarkers

"Diabetes Associated Proteins Database (DAPD)" has been developed to link the diabetes

associated genes, pathways and proteins. The current version of DAPD has been built with

proteins associated with different types of diabetes. DAPD is open accessed via following URL:

www.mkarthikeyan.bioinfoau.org/dapd.

(1) Diabetes mellitus

References Diabetes type Proteomics platform

(Alkhalaf et al., 2010) 2 CE-MS

(Hsu et al., 2015) DN MS-based proteomics

(Sung et al., 2015) Diabetes complications MS –based proteomics

(Vujosevic et al., 2015) Diabetic retinopathy MS-based proteomics

(Preil et al., 2015) Cardiovascular diseases

associated with type 2

diabetes

LC/MS/MS

(Bhatt et al., 2015) 1 LC/MS/MS

Proteomics/diabetes studies

Malignancies

Therapy, Diagnosis and Prognosis

Genetic

instability

Impact of

progression

Impact of

prognosis

impact of

genesis

PROTEOMICS/ONCOPROTEOMICS

Proteomics/malignancies studies

Data search for proteomics/ malignancies

Oncoproteomics

(1) Leukemias

References Proteomics/Leukemias

(Baczek, 2014). Early diagnosis of leukemias

(Roboz and Roboz, 2015). Identification of therapeutic targets

Identification of biomarkers, drug discovery for leukemias

(Lopez Villar et al., 2015) Early diagnosis, management and development of

personalized treatment of acute lymphoblastic leukaemia (ALL)

(Aasebo et al., 2015) Identification of protein biomarkers in acute myeloid leukemia (AML)

(Priola et al., 2015). Studying the changes in CSF proteome in patients with

ALL

(Singh et al., 2015) Diagnosis of chronic lymphocytic leukemia (CLL)

(2) Gastrointestinal cancers

References Gastrointestinal cancers/proteomics

(Subbannayya et al., 2015). Many proteins show alterations in gasteric cancers.

(Shevchenko et al., 2014). Low molecular weight proteins detected as a strong markers for gasteric cancer.

(Abramowicz et al., 2015). Differentiation between locally advanced and metastatic cancer types.

(Subbannayya et al., 2015) Proteins markers for gasteric cancer.

(Lee et al., 2014). Complement factor b (CFB) as a candidate

serologic biomarker for pancreatic cancer diagnosis.

References Data obtained

(Dun et al., 2015) Identification of Potential Biomarkers and Therapeutic

Targets for Brain Metastasis.

(Thomas et al., 2015) Detection of proteomes with specific functional

significance in breast cancer.

(Sjostrom et al., 2015) Identification of proteins as potential biomarkers for

breast cancer recurrence.

(Lawrence et al., 2015) The genome and proteome complementary information

as powerful engine for therapeutic discovery.

(Edwards et al., 2015) Proteomic investigations of breast cancer.

(Milioli et al., 2015) Comparative proteomics of primary breast carcinomas

and lymph node metastases.

(Kern et al., 2015) Identification of Proteins in normal and malignant

mammary epithelial cells.

(3) Breast cancer

(4) Hepatocellular carcinoma

References Data obtained

(Reis et al., 2015) Novel and reliable protein biomarker in panel based

differential diagnostics of liver tumors.

(Goncalves Lda et al., 2014) Compare the salivary proteome to detect HCV.

(Tit-Oon et al., 2014) Comparative secretome analysis of

cholangiocarcinoma.

(Yin et al., 2015) Screen the changes in site-specific proteins in cirrhosis

patients.

(Borlak et al., 2015) Detect novel molecular pathogenesis liver cancer

(Shen et al., 2015; Zhang et

al., 2015a).

Detect Transcripted proteins in metastatic and non

metastatic liver cancers.

(Shao et al., 2015). Investigation of cirrhosis (CIR) and HCC based on a

urinary proteomics techniques.

3- Protein net work mapping

1. Proteins “work together” by actually binding to form multicomponent

complexes that carry out specific functions (protein-protein interaction)

3. Biochemists have come to appreciate that essentially all proteins bind to

or interact with at least one other protein.

Advantages of Protein network mapping

4- Mapping of protein modifications

1. Finding all modifications on a single protein Identified by examining the

measured mass and fragmentation spectra.

2. Proteome wide scanning of modifications.

The Future of Proteomics

Great potential for medical and

biological advances.

Rapid analysis of thousands of unique

proteins.

Complement to genome data.

1. Disease diagnostics.

2. Rationally designed drugs.

Aaser Abdelazim