1. Protein (Identification, Expression, Purification)

2. Mass spectrometry

Biotechnology

http://facultymembers.sbu.ac.ir/mirzajani/courses/

Amino acids are biologically important organic compounds composed of amine (-NH2) and carboxylic acid (-COOH) functional groups, along with a side-chain specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, though other elements are found in the side-chains of certain amino acids.

They can be classified according to the core structural functional groups' locations as alpha- (α-), beta- (β-), gamma- (γ-) or delta- (δ-) amino acids; other categories relate to polarity, pH level, and side-chain group type (aliphatic, acyclic, aromatic, containing hydroxyl or sulfur, etc.).

INTRODUCTION : Amino acids

amine carboxylic acid

• Capacity to polymerize

• Useful acid-base properties

• Varied physical properties

• Varied chemical functionality

http://en.wikipedia.org/wiki/Biologyhttp://en.wikipedia.org/wiki/Organic_compoundhttp://en.wikipedia.org/wiki/Aminehttp://en.wikipedia.org/wiki/Carboxylic_acidhttp://en.wikipedia.org/wiki/Functional_grouphttp://en.wikipedia.org/wiki/Side-chainhttp://en.wikipedia.org/wiki/Side-chainhttp://en.wikipedia.org/wiki/Side-chainhttp://en.wikipedia.org/wiki/Acidhttp://en.wikipedia.org/wiki/Carbonhttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Nitrogenhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Alpha_and_beta_carbonhttp://en.wikipedia.org/wiki/Chemical_polarityhttp://en.wikipedia.org/wiki/PHhttp://en.wikipedia.org/wiki/Aliphatichttp://en.wikipedia.org/wiki/Open-chain_compoundhttp://en.wikipedia.org/wiki/Aromatichttp://en.wikipedia.org/wiki/Sulfur

NH2

C

(H3C)

2HC

H

NH2

C COOHH

CH9(CH

3)2

(S) -Enantiomer (R) -Enantiomer

Draw tetrahedral 3D structures for (R) and (S) valine

HOOC

Amino Acids: Classification

Common amino acids can be placed in five

basic groups depending on their R substituents:

• Nonpolar, aliphatic (7)

• Aromatic (3)

• Polar, uncharged (5)

• Positively charged (3)

• Negatively charged (2)

Aliphatic Amino Acids

Aromatic Amino Acids

Charged Amino Acids

Polar Amino Acids

Special Amino Acids

aa are high melting point solids! Why?

Answer = aa are ionic compounds under normal conditions

C

O

OHR

NH3

C

O

OR

NH3

C

O

OR

NH2

LOW pH

Zwitterion

NEUTRAL

Carboxylate Form

HIGH pH

ammonium Form

Isoelectric Point = concentration of zwitterion is at a maximum and the concentration of cations and anions is equal

For aa with basic R-groups, we require higher pHs, and for aa with acidic R-groups, we require lower pHs

to reach the Isoelectric Point

INTRODUCTION : Peptides Peptides are short chains of amino acid monomers linked by peptide (amide) bonds.

1 2,3 4 The shortest peptides are dipeptides, consisting of 2 amino acids joined by a single peptide bond, followed by tripeptides, tetrapeptides, etc.

A polypeptide is a long, continuous, and unbranched peptide chain. Hence, peptides fall under the broad chemical classes of biological oligomers and polymers, alongside nucleic acids, oligo- and polysaccharides, etc.

Amino acids that have been incorporated into peptides are termed "residues" due to the release of either a hydrogen ion from the amine end or a hydroxyl ion from the carboxyl end, or both, as a water molecule is released during formation of each amide bond. All peptides except cyclic peptides have an N-terminal and C-terminal residue at the end of the peptide (as shown for the tetrapeptide in the image).

N-terminal C-terminal

http://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Monomerhttp://en.wikipedia.org/wiki/Peptide_bondhttp://en.wikipedia.org/wiki/Amidehttp://en.wikipedia.org/wiki/Dipeptidehttp://en.wikipedia.org/wiki/Tripeptidehttp://en.wikipedia.org/wiki/Tetrapeptidehttp://en.wikipedia.org/wiki/Polypeptidehttp://en.wikipedia.org/wiki/Oligomerhttp://en.wikipedia.org/wiki/Polymerhttp://en.wikipedia.org/wiki/Nucleic_acidhttp://en.wikipedia.org/wiki/Polysaccharidehttp://en.wikipedia.org/wiki/Cyclic_peptidehttp://en.wikipedia.org/wiki/N-terminushttp://en.wikipedia.org/wiki/N-terminushttp://en.wikipedia.org/wiki/N-terminushttp://en.wikipedia.org/wiki/C-terminushttp://en.wikipedia.org/wiki/C-terminushttp://en.wikipedia.org/wiki/C-terminus

H2O

Amino Acid 2

N

R

COOH

C

H

H

H

Amino Acid 1

NH2

R

C C

H

O

OH

Peptide bonds form by dehydration synthesis

Peptide classes 1. Milk peptides

Two naturally occurring milk peptides are formed from the milk protein casein when digestive enzymes break this down; they can also arise from the proteinases formed by lactobacilli during the fermentation of milk.

2. Ribosomal peptides Ribosomal peptides are synthesized by translation of mRNA. They are often subjected to proteolysis to generate the mature form. These function, typically in higher organisms, as hormones and signaling molecules.

3. Nonribosomal peptides These peptides are assembled by enzymes that are specific to each peptide, rather than by the ribosome. The most common non-ribosomal peptide is glutathione, which is a component of the antioxidant defenses of most aerobic organisms. Other nonribosomal peptides are most common in unicellular organisms, plants, and fungi and are synthesized by modular enzyme complexes called nonribosomal peptide synthetases.

4. Peptones Peptones are derived from animal milk or meat digested by proteolysis. In addition to containing small peptides, the resulting spray-dried material

5. Peptide fragments Peptide fragments refer to fragments of proteins that are used to identify or quantify the source protein. Often these are the products of enzymatic degradation performed in the laboratory on a controlled sample, but can also be forensic or paleontological samples that have been degraded by natural effects.

NTRODUCTION :Proteins Proteins are large biological molecules, or macromolecules, consisting of one or more chains of amino acid residues. Proteins perform a vast array of functions within living organisms, including : 1. catalyzing metabolic reactions, 2. replicating DNA, 3. responding to stimuli, and 4. transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in folding of the protein into a specific three-dimensional structure that determines its activity.

Proteins are:

• Cofactor is a general term for functional non-amino acid component – Metal ions or organic molecules

• Coenzyme is used to designate an organic cofactors – NAD+ in lactate dehydrogenase

• Prosthetic groups are covalently attached cofactors – Heme in myoglobin

• Polypeptides (covalently linked -amino acids) + possibly –

• cofactors,

• coenzymes,

• prosthetic groups,

• other modifications

19

Classification of Proteins

• Simple: hydrolyze to amino acids only.

• Conjugated: bonded to a nonprotein group, such as sugar, nucleic acid, or lipid.

• Fibrous: long, stringy filaments, insoluble in water, function as structure.

• Globular: folded into spherical shape, function as enzymes, hormones, or transport proteins.

20

Levels of Protein Structure

• Primary: the sequence of the amino acids in the chain and the disulfide links.

• Secondary: structure formed by hydrogen bonding. Examples are -helix and pleated sheet.

• Tertiary: complete 3-D conformation.

• Quaternary: association of two or more peptide chains to form protein.

Chapter 24 24

Pleated Sheet Each carbonyl oxygen hydrogen bonds with an N-H hydrogen on an adjacent peptide chain.

=>

QUANTIFICATION METHODS:

2.1. Fluorescent Assays

2.1.1. Fluoro Profile

2.2. Colorimetric Assays

2.2.1. BCA

2.2.2. Bradford

2.2.3. Lowry

2.2.4. Micro Pyrogallol Red Method

26

2.1. Fluorescent Assays: 2.1.1. Fluoro Profile The FPQ Kit is significantly more sensitive than existing standard colorimetric measurements (Bradford and Bicinchoninic acid assays (BCA)) and exhibits a larger linear dynamic range than other fluorimetric protein determination kits.

FluoroProfile uses the water-soluble fluorophore epicocconone allowing for safe economical disposal. Results are achieved in 20 minutes and reversible binding of the fluorophore allows for mass spectrometry and other downstream assays.

Features and Benefits:

• Sensitive – Detects over three orders of magnitude down to 40 ng/ml

• Simple – Results available in 20 minutes and stable for six hours

• Safe – Water soluble, biodegradable fluorophore enables safe economical disposal

• Yields an intense fluorescent red on protein binding

• Binding is completely reversible making the protein suitable for mass spectrometry and functional assays

• Water soluble proteins may be quantified without using organic solvents

• A large dynamic range, large Stokes shift and highly sensitive

• Highly tolerant of substances commonly interfering with protein quantification

• Biodegradable and environmentally friendly

27

2.1.1. Fluoro Profile

Components:

• FluoroProfile Fluorescent Reagent, 10 ml

• Quantification Buffer, 10 ml

• BSA Standard, 2 mg

Mackintosh JA et al. Fluoroprofile, a fluorescence-based assay for rapid and sensitive quantitation of proteins in solution. Proteomics. 2005;5(18):4673-7.

There is a concomitant shift in the emission maximum from 520 to 605 nm after binding, which results in low background signal allowing a linear dynamic range of 40 ng/mL to 200 μg/mL for most proteins.

shipped in wet ice

storage temp. −20°C

Epicocconone represents a new class of natural fluorescent probes based on a polyketide skeleton isolated from the fungus Epicoccum nigrum. Epicocconone is a small, cell permeable natural product with a high molar absorbtivity and a long Stokes' shift that will be useful in biotechnological applications.

28

Lecture information/FluoroProfile protocol.pdf

2.2. Colorimetric Assays: 2.2.1. BCA Protein determination is one of the most common operations performed in biochemical research. The principle of the bicinchoninic acid (BCA) assay is similar to the Lowry procedure, in that both rely on the formation of a Cu2+-protein complex under alkaline conditions, followed by reduction of the Cu2+ to Cu1+. The amount of reduction is proportional to the protein present. It has been shown that cysteine, tryptophan, tyrosine, and the peptide bond are able to reduce Cu2+ to Cu1+. BCA forms a purple-blue complex with Cu1+ in alkaline environments, thus providing a basis to monitor the reduction of alkaline Cu2+ by proteins. The BCA assay is more sensitive and applicable than either biuret or Lowry procedures. In addition, it has less variability than the Bradford assay.

The BCA assay has many advantages over other protein determination techniques: • The color complex is stable • There is less susceptibility to detergents • It is applicable over a broad range of protein concentrations 29

2.2. Colorimetric Assays: 2.2.1. BCA 200-1000 µg/ml protein Proteins reduce alkaline Cu(II) to Cu(I) in a concentration-dependent manner. Bicinchoninic acid is a highly specific chromogenic reagent for Cu(I), forming a purple complex with an absorbance maximum at 562 nm. The absorbance is directly proportional to protein concentration.

Components: 1. Bicinchoninic Acid Solution 2. 4%(w/v) CuSO4 • 5H2O Solution 3. BSA Protein Standard

http://www.piercenet.com/method/chemistry-protein-assays 30

2.2. Colorimetric Assays: 2.2.2. Bradford

The Bradford Reagent, can be used to determine the concentration of proteins in solution. The procedure is based on the formation of a complex between the dye, Brilliant Blue G, and proteins in solution. The protein-dye complex causes a shift in the absorption maximum of the dye from 465 to 595 nm. The amount of absorption is proportional to the protein present. The Bradford Reagent requires no dilution and is suitable for micro, multiwell plate, and standard assays. The linear concentration range is 0.1-1.4 mg/ml of protein, using BSA (bovine serum albumin) as the standard protein.

31

2.2. Colorimetric Assays: 2.2.2. Bradford

Features and Benefits 1. The reagent is ready to use. No mixing or dilution required. 2. Color development is rapid. Only a five minute incubation and then the sample is

read a 595 nm. 3. Reducing sugars and reducing substances along with thiols do not interfere with this

reagent. 4. Reagent is suitable for micro (1-10 μg/ml) and standard (50-1400 μg/ml) assays. 5. Can be used in microwell plate assays. 6. Inexpensive assay.

storage temp. 2-8°C

Advantages: The Bradford Reagent is compatible with reducing agents, which are often used to stabilize proteins in solution. Other protein assay procedures (Lowry and BCA) are not compatible with reducing agents. So, the Bradford Reagent should be used in place of these protein assays if reducing agents are present.

Disadvantage: However, the Bradford Reagent is only compatible with low concentrations of detergents. If the protein sample to be assayed has detergents present in the buffer, it is suggested to use the BCA protein determination procedure. 32

2.2. Colorimetric Assays: 2.2.3. Lowry

33

The Lowry Assay, is a common method for quantitation of soluble protein. Due to its sensitivity, simplicity, and precision, it is often a method of choice. Based on two chemical reactions

1. in which alkaline cupric tartrate complexes with the peptide bond of the protein and

2. a reduction with Folin and Ciocalteu's reagent. This reaction yields a blue-purple color in which the absorption is read between 500 and 800 nm. This assay may be performed directly with a protein solution, or a precipitation method involving DOC (deoxycholic acid) and TCA (trichloroacetic acid) may be used. Precipitation eliminates interference often caused by other reagents such as Tris, ammonium sulfate, EDTA, sucrose, citrate, and others.

Peterson, G.L., Analyt. Biochem., 83, 346 (1977).

2.2. Colorimetric Assays: 2.2.3. Lowry

34

Components: • Lowry Reagent, Powder • 0.15% Deoxycholate (DOC) Solution • Trichloroacetic Acid (TCA) Solution • Folin and Ciocalteu's Phenol Reagent • BSA Standard, 2 mg

The method combines the reactions of copper ions with the peptide bonds under alkaline conditions) with the oxidation of aromatic protein residues. The Lowry method is best used with protein concentrations of 0.01–1.0 mg/mL and is based on the reaction of Cu+, produced by the oxidation of peptide bonds, with Folin–Ciocalteu reagent.

The reaction mechanism is not well understood, but involves reduction of the Folin reagent and oxidation of aromatic residues (mainly tryptophan, also tyrosine). • Experiments have shown that cysteine is also reactive to the reagent. Therefore, cysteine residues

in protein probably also contribute to the absorbance seen in the Lowry Assay.

The concentration of the reduced Folin reagent is measured by absorbance at 750 nm. As a result, the total concentration of protein in the sample can be deduced from the concentration of Trp and Tyr residues that reduce the Folin reagent.

http://en.wikipedia.org/wiki/Peptide_bondhttp://en.wikipedia.org/wiki/Alkalinehttp://en.wikipedia.org/wiki/Oxidationhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Folin%E2%80%93Ciocalteu_reagenthttp://en.wikipedia.org/wiki/Folin%E2%80%93Ciocalteu_reagenthttp://en.wikipedia.org/wiki/Folin%E2%80%93Ciocalteu_reagenthttp://en.wikipedia.org/wiki/Folin%E2%80%93Ciocalteu_reagenthttp://en.wikipedia.org/wiki/Folin%E2%80%93Ciocalteu_reagenthttp://en.wikipedia.org/wiki/Aromatichttp://en.wikipedia.org/wiki/Tryptophanhttp://en.wikipedia.org/wiki/Tyrosine

2.2. Colorimetric Assays: 2.2.4. Micro Pyrogallol Red Method

35

Sigma's micro pyrogallol red procedure is a modification of a published method. The method is based on measuring the shift in the absorption that occurs when the pyrogallol red molybdate complex binds to basic amino acid groups of protein molecules. The increase in absorbance at 600 nm is directly proportional to protein concentration in the sample. This method displays a linear range from 0.01-2.0 mg/ml.

Fujita, Y., et al., Color reaction between pyrogallol red-molybdenum (VI) complex and protein. (abstract) Bunseki Kagaku, 32, E379–386 (1983) - See more at: http://www.sigmaaldrich.com/life-science/proteomics/protein-quantitation/tools-for-protein.html#fluoroprofile

36 Fujita, Y., et al., Color reaction between pyrogallol red-molybdenum (VI) complex and protein. (abstract) Bunseki Kagaku, 32, E379–386 (1983) - See more at: http://www.sigmaaldrich.com/life-science/proteomics/protein-quantitation/tools-for-protein.html#fluoroprofile

Components: • Total Protein Reagent • Protein Standard Solution; Human Serum Albumin

usage sufficient for ~240 manual assays

storage temp. 2-8°C

2.2. Colorimetric Assays: 2.2.4. Micro Pyrogallol Red Method

3. QUALIFICATION METHODS:

37

why you need to separate and quality study of proteins?

Catalysis: Enzymes – enolase (in the glycolytic pathway)

– DNA polymerase (in DNA replication)

Transport: – hemoglobin (transports O2 in the blood)

– lactose permease (transports lactose across the cell membrane)

Structure: – collagen (connective tissue)

– keratin (hair, nails, feathers, horns)

Motion: – myosin (muscle tissue)

– actin (muscle tissue, cell motility)

Gentle lysis methods

38

Pre

cip

itat

ion

pro

ced

ure

s

39

Removal of contaminants

40

Removal of contaminants

41

Removal of contaminants

42

Electrophoresis for Protein Analysis

Separation in analytical scale is commonly done by electrophoresis

– Electric field pulls proteins according to their charge

– Gel matrix hinders mobility of proteins according to their size and shape

SDS PAGE: Molecular Weight

• SDS – sodium dodecyl

sulfate – a detergent

• SDS micelles binds to,

and unfold all the

proteins

– SDS gives all proteins an

uniformly negative

charge

– The native shape of

proteins does not matter

– Rate of movement will

only depend on size:

small proteins will move

faster

-

3. QUALIFICATION METHODS: 3.1. Gel electrophoresis Separation base method

45

What is Electrophoresis ?

The migration of a charged particle (protein) through a separation matrix toward the opposite charged electrode

+ _

_ _ _ _ + + + ++ _

1st Dimension

2ed Dimension

Separate native proteins by size – proteins stop moving when they reach a sertain gel density (but this may take a very long time ...)

A great technique to study protien oligomerization!

3.1. Gel electrophoresis

So, ...

3.1. Gel electrophoresis Please notice to:

48

200

200

200 200

45

45

45

45 45

6.5

200

3.1. Gel electrophoresis Please notice to:

49

3.1. Gel electrophoresis: Sample preparation

Optional modifications for more proteins displayed, shorter focusing time, and more sharply focused spots:

- Chaotropes: - 8M Urea

- 2M Thiourea & 7M Urea

- Surfactants: - 4% CHAPS

- 2% CHAPS & 2% Sulfobetaine 3-10

- Reducing agents: - 100mM DTT

- 2mM TBP (Tributyl phosphine) and/or 65mM DTT

50

3.1. Gel electrophoresis: Sample preparation

52

Protein Solubilization

Increases the solubility of some proteins 2-20 mM Tris base (Carrier ampholytic buffer)

5-20 mM DTT (to reduce disulfide bonds) 8 M Urea (neutral chaotrope)

Chaotropic agents interfere with stabilizing non-covalent forces

(hydrogen bonds, van der Waals forces, and hydrophobic)

4% CHAPS Detergent (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate)

– pH of 5-7 – Zwitterionic detergent (electronically neutral-has a both Neg and Pos

useful for varible charged peptides ) – Protects the native state of proteins – Better when downstream apps include IEF because no affect on pH

gradients

IEF and IPG (immobilized pH Gradient) • Strip of paper Made by covalently integrating acrylamide

and variable pH ampholytes • Separation on basis of pI, not MW • Available in different pH ranges

Requires very high voltages (5000V)and long period of time (10h)

55

SDS Gel

IPG strip-pressed down into the SDS-PAGE gel

Positive electrode

Negative electrode

Similar mw but different pI

Similar pI but different mw

pH 3 4 5 6 7 8 9 10

3.1. Gel electrophoresis: Staining

57

Stain Sensitivity (ng/spot) Advantages

Coomassie-type 5-10 Simple, fast

Silver stain 1-4 Very sensitive, laborious

Copper stain 5-15 Reversible, 1 reagent

negative stain

Zinc stain 5-15 Reversible, simple, fast

high contrast neg. stain

SYPRO ruby 1-10 Very sensitive, fluorescent

3.1. Gel electrophoresis: Staining

Staining Polyacrylamide Gels Coomassie Blue Stain- can usually detect a 10-50 ng protein per band

Blue Safe Stains -Similar to Coomassie. Destaining optional or water rinse. (8 ng/band)

BioSafe Blue

SimplyBlue

GelCode

InstantBlue-destaining not recommended

Silver Staining- 50 times more sensitive than Coomassie Blue. (0.3ng/BAND)

Fixation [Acetic acid-methanol]

Sensitize gel with sodium thiosulfate

Stain with silver solution

Rinse with water

Develop with formaldehyde and carbonate followed by stopping with Glacial acetic

Fluoresecnt Stains –almost as sensitive as Silver but requires excitation source

Flamingo Fluorescent Gel Stain

Deep Purple* Total Protein Stain

SYPRO* Ruby Protein Gel Stain

Krypton Protein Stain

IR stains

Coomassie –Protein Binding

Sulfonic acid group interacts with positively charged amine R groups

Basic amino acids including arginine, lysine and histidine but weakly with histidine, tyrosine, tryptophan and phenylalanine

Interactions in its anionic form [-]

Electrostatic

Ionic

Van der Waals

Hydrophobic

Destaining Gels Most gels require destaining to see banding and to eliminate background stain for high resolution.

Gels with abundant protein need not be destained when using certain SafeBlue stains such as InstantBlue

Coomassie blue destaining

Usually requires acetic acid , methanol, and water

Safe Blue destaining

Usually requires water rinse

Silver Stains

1. Some methods use Potassium Ferricynide -Sodium Thiosulfate solutions 2. Some methods use Sodium chloride -Cupric sulfate -Sodium thiosulfate pentahydrate. 3. Some destaining may require a stop solution including 10% Acetic acid

2D Gel Post Analysis

Compare gel images and determine what bands/spots are different Requires software to compare gels

Apparent difference- Need to extract spot for MS

Lecture information/2D Electrophoresis.pdf

Chemical Identification Comparison of Physical Properties Boiling Point, Melting Point, Density, Optical

rotation, Appearance, Odor

Elemental Analysis Burn the compound and measure the amounts of CO2, H2O and other components that are produced to determine the empirical formula

Spectroscopic Methods for Structure Determination Ultraviolet-Visible (UV/Vis) spectroscopy:

determination of solutions of transition metal ions and highly

conjugated organic compounds

Infrared (IR) spectroscopy:

Functional groups

Mass spectrometry (MS):

Molecular mass and formula and structure information

Nuclear magnetic resonance (NMR) spectroscopy:

Map of carbon-hydrogen framework

What is MS?

• Mass spectrometry (MS) :

• An analytical technique by using mass spectrometry for the determination of the composition of a sample or molecule and elucidation of the chemical structures of molecules, such as peptides/proteins and other chemical compounds.

• Mass spectrometry has been described as the smallest scale in the world, not because of the mass spectrometer’s size but because of the size of what it weighs -- molecules.

Mass spectrometry (Mass Spec or MS) uses high energy

electrons/photon to ionize/break a molecule into fragments.

How MS works?

Separation and analysis of the fragments provides information about:

– Molecular weight

– Structure

8 9 10 11 12 13 14 15 16

mass N

um

be

r o

f co

un

ts

+

e- e-

e-

heavy

light + A

+

B

+

C

A+ B+ C+

How MS works?

The MS process: sample inlet

• Gas Inlet: CO2 ,N2 ,C6H6, volatile liquids

• Solids Probe: Heat to 250°C to vaporize sample, Cholesterol, triglycerides, drugs, many natural products

• Gas or Liquid Chromatography: Mixture analysis Plant extracts, biological fluids, environmental samples Easily automated

• Direct Liquid Injection: Fast and easily automated (1 sample/min) Auto injection

• Spotting on a sample plate: Very high throughput

Why we need MS?

Information is composition-specific – Very selective analytical technique

– Most other spectroscopies can describe functionalities, but not chemical formulae

MS is VERY sensitive – mg/L to pg/L sensitivity possible, Picomole sensitivity is common

in the MS

Mass spectrometers have become MUCH easier to use in the last 15 years

Molecular weight is an intrinsic property of a substance

Mass spectrometry is readily coupled to chromatographic techniques.

What information can be found?

Molecular weight

Molecular formula (HRMS)

Structure (from fragmentation fingerprint)

Isotopic incorporation / distribution

Protein sequence (MS-MS)

MS dictionary …..

• Nominal molecular weight: Calculated using the nominal (non fractional) atomic weight for the most abundant isotope (which for many elements is also the lowest mass isotope; (H = 1, C = 12, O = 16, Cl = 35).

• Monoisotopic exact molecular weight: Calculated using the exact atomic weight for the most abundant isotope; (H = 1.00782, C = 12.0000, O = 15.9949, Cl = 34.9689).

• Average exact molecular weight: Calculated from the exact atomic weights of all of the isotopes, each isotope weighted according to its abundance (H = 1.00794, C = 12.0107,O = 15.9994, Cl = 35.4527).

MS readily differentiates isotopes, which have different atomic mass

units (amu) or Daltons (Da).

MS dictionary ….. Isotope Peaks: consider the probability that a molecule has 0, 1, 2… heavy isotopes.

Selected Ion Monitoring (SIM):

Plot intensity of a particular ion vs

Time. Used most often with GC and LC.

Total Ion Current plot (TIC):

Plot of signal for ions of all m/z vs time.

Example …

MS dictionary …..

Isotopic abundances: 12C = 98.93%, 13C =1.07%, 1H = 99+%, 2H = 0.012%

MS dictionary ….. • Molecular ion & Fragmentation

• A “soft” and “hard” ion source.

• Isotopic ratios: For example the M+1 peak provides information on elemental composition.

• m is the nominal mass of the 1st peak

• Δm is the mass difference between the 2 peaks

• Mass analyzers resolution is 500,ooo.

• If it resolve 200.00 and 200.01

• So RP is 200.00/0.05=4000

MS dictionary …..

• Decay Vs. Fragmentation:

• Decay: – 1- ISD: in source decay

– 2- PSD: post source decay

• Fragmentation: – 1- Parental ion

– 2- Daughter ion

Units 1 Da = 1 u = 1.6605*10-27 kg (1/12 of a 12C atom) 1 Th = 1 Da/e = 1.0364*10-8 kg/C

Molecular Weight Calculations

The molecular weight is computed by summing the masses

of all atoms in the compound/ion.

Erythromycin (M+H)+: C37H68N1O13+ = 12.011*37 + 1.008*67 +

14.007 + 15.999*13 = 734.93 Da

Yet 734.5 is observed by ESI-MS

O

O

O

O

O

OH

O

NH+HO

O

OH

OHHO

http://upload.wikimedia.org/wikipedia/commons/d/d5/Erythromycin_3d_structure.png

Applications … Sample preparation in mass spectrometry

The sample preparation for mass

spectrometry is used for the optimisation

of a sample for the analysis in a mass

spectrometer (MS). These have depending

on their ion source different requirements

for volume, concentration, and

composition of the analyte solution.

Furthermore, in protein mass

spectrometry usually the protein of

interest is cleaved into peptides before

analysis, either by in-gel digestion or

by proteolysis in solution.

Sample Preparation

Software Instrument

2. Instrumentation:

2.1. Ion Sources

EI, CI, FD, SIMS, FAB, MALDI, APCI, APLI, TSP, ESI, ICP.

2.2. Analyzers

Sector, Time-of-flight, Quadrupole mass filter, Quadrupole ion

trap, FT-ICR, Orbitrap and Hybrid methods.

2.3. Detectors

Electron multiplier, Microchannel plate detector, Daly detector,

Faraday cup.

• Matrix-Assisted Laser Desorption Ionization

2.1. Ion Sources: MALDI

Applications … Sample preparation in mass

spectrometry (MALDI)

A separate desalting step is usually not necessary as in previous steps of sample

preparation e.g. in-gel digestion of proteins, the selection of appropriate buffer salts

prevents the occurrence of this problem. Furthermore the crystallised matrix/analyte

mixture allows removing of salts by washing on the target and the addition

of ammonium phosphate before crystallisation can improve the signal quality.

MALDI matrices used for identifying peptides include CHCA and DHB. These

solutions are typically saturated solutions in a 50% Acetonitrile/0.1% TFA; TFA

being the protonating agent. Typically the sample solution and matrix solution is

mixed in a 1:1 ratio and then a half a microliter to a microliter is added to the MALDI

plate.

For matrix-assisted laser

desorption/ionization (MALDI) mass

spectrometry the sample is usually mixed with

a matrix solution and spotted on the target

plate. The matrix crystallises together with the

sample and the analyte molecules are transferred

to the gas phase by the pulsed laser irradiation.

2.1. MALDI: sample injection

2.1. MALDI: influential parameters

LASER

2.1. MALDI: influential parameters

MATRIX

MALDI properties

Good solubility

Vapour pressure must be sufficiently low to maintain vacuum

conditions

Viscosity must allow diffusion of the analyte from the bulk to the

surface

Polar : to solvate and separate preformed ion

Less Sensitive to Salts

Lower PRACTICAL detection limits

Easier to interpret spectra (less multiple charges)

Quick and easy

Higher mass detection

Higher Throughput (>1000 samples per hour)

2.1. Ion Sources: ESI

• Electrospray ionization (ESI) is a technique used in mass spectrometry to produce ions. It is especially useful in producing ions from macromolecules because it overcomes the propensity of these molecules to fragment when ionized.

http://en.wikipedia.org/wiki/File:NanoESIFT.jpg

ESI properties . . .

Advantages

Electrospray Ionization can be

easily interfaced to LC.

Absolute signals from

Electrospray are more easily

reproduced, therefore, better

quantitation.

Mass Accuracy is considered better.

Multiple charging is more

common then MALDI.

Disadvantages

No Fragmentation

Need Polar Sample

Need Solubility in Polar Solvent (MeOH, ACN, H2O, Acetone are best)

Sensitive to Salts

Suppression

ESI: concentration effect

ESI: molecular weight effect

Consider a peptide with MW of 10000 With ESI-MS, charges by H+

addition

M + nH+ MnHn+

Resultant ions formed are :-

When z = 1 m/z = (10000+1)/1 = 10001

When z = 2 m/z = (10000+2)/2 = 5002

When z = 3 m/z = (10000+3)/3 = 3334.3

When z = 4 m/z = (10000+4)/4 = 2501

When z = 5 m/z = (10000+5)/5 = 2001

Multiple Charging

ESI: some improvements

ESI: some improvements

ESI: some improvements

2. Instrumentation:

2.1. Ion Sources

EI, CI, FD, SIMS, FAB, MALDI, APCI, APLI, TSP, ESI, ICP.

2.2. Analyzers

Sector, Time-of-flight, Quadrupole mass filter, Quadrupole ion

trap, FT-ICR, Orbitrap.

2.3. Detectors

Electron multiplier, Microchannel plate detector, Daly detector,

Faraday cup.

2.2. Analyzers:

2.2. Analyzers: TOF

• Time-of-flight mass spectrometry (TOFMS) is a method of mass spectrometry in which an ion's mass-to-charge ratio is determined via a time measurement. Ions are accelerated by an electric field of known strength.This acceleration results in an ion having the same kinetic energy as any other ion that has the same charge. The velocity of the ion depends on the mass-to-charge ratio.

TOF … • The LTOF analyser separates ions, after their initial acceleration by an electric field, according to their velocities when they drift in a free-field region that is called a flight tube.

• The RTOF creates a retarding field that acts as an ion mirror by deflecting the ions and sending them back through the flight tube. It can be single stage or multiple stage.

• In the oaTOF, orthogonal acceleration gives a new component of velocity to the ions that are in the ion beam. This component is orthogonal and thus independent of the velocity of the ions in the beam.

RTOF …

In this method, the optimum reflectron potential for observing the precursor ion is not the optimum for the fragment ions, and must be set up separately for each fragment ion. Indeed, if the reflectron potential is optimized for the precursor ion, the fragment ions are not focused on the detector.

TOF properties . . .

Advantages:

Good for kinetic studies of fast reactions and for use with gas chromatography to analyze peaks from chromatograph

High ion transmission

Can register molecular ions that decompose in the flight tube

Extremely high mass range (>1MDa)

Fastest scanning

Disadvantages:

Requires pulsed ionization method or ion beam switching (duty cycle is a factor)

Low resolution (4000)

Limited precursor-ion selectivity for most MS/MS experiments

2.2. Analyzers: FT-ICR

2.2. Analyzers: FT-ICR

• Ion Cyclotron Resonance and Fourier Transform the ions are injected into a box a few centimetres along its side, located in a magnetic field of 3 to 9.4 T produced by a superconducting magnet. The relationship between the frequency and the mass shows that determining the mass in this case consists of determining the frequency. The latter can be measured according to several methods which are classified into one of two categories: those based on observing isolated frequencies and those using complex waves and Fourier transforms.

FT-ICR … function of time is transformed, through a Fourier transform, into intensity as a

function of frequency

ICR properties … Advantages

The highest recorded mass resolution of all mass spectrometers (>500,000)

Very good accuracy (

….MALDI or Electrospray ?

MALDI is limited to solid state, ESI to liquid

ESI is better for the analysis of complex mixture as it is directly

interfaced to a separation techniques (i.e. HPLC or CE)

MALDI is more “flexible” (MW from 200 to 400,000 Da)