Proteomics and biochemical methodologies

Proteomics and biochemical methodologies

25/02/19 Program

Proteomics: technologies used to characterize the proteome: methodologies, purification techniques and MS and

bioinformatics.

Methods of separation of proteins used in proteomics:

- Bi-dimensional electrophoresis (2D-PAGE).

- Orthogonal chromatography (Mud-PIT).

Methods of identification:

- Classical protein sequencing methods by Edman degradation.

- Mass spectroscopy (MS):

Theory: physical principles governing mass separation.

o Instruments: samples application, ion sources, analyzers, detectors.

o MS analysis: determination of the molecular weight.

o MS-MS analysis (or tandem MS): determination of the structure.

o Interpretation of the MS and MS-MS spectra.

o

Applications in proteomics:

- Post-translational modifications (PTMs) of proteins.

- Quantitative proteomics.

- Functional proteomics.

- Diagnostic proteomics. Introduction: proteome and proteomics

Proteome: “proteins expressed by a genome”, set of proteins present in a cell, tissue, organism at a certain time

point.

Genomics deal with the discovery of all the DNA sequences in the entire genome. The genome is the complete set

of genes inside a cell. The initial product of genome expression is the transcriptome: collection of RNA molecules

derived from those protein-coding genes whose biological information is required by the cell at a particular time.

Proteomics deals with the large-scale determination of gene and cellular function directly at the protein level, the

proteome.

Genomics: techniques for identification of genes, discovery of new genes, determination of genes sequences, …

Transcriptomics: techniques for information on gene expression in specific tissue, on the relation between gene

activity and cellular function, under physiological conditions or upon external stimuli.

Proteomics: characterization of proteins population in a cell or a tissue, in term of localization, modifications,

interaction and turn-over, under physiological conditions upon external stimuli.

Human genome project: complete mapping and understanding of all the genes of human beings (≈20k).

Human proteomic project: map of the protein-based molecular architecture of the human body and a resource to

elucidate biological and molecular function and advance diagnosis and treatment of diseases.

· Features of proteome

Complexity

Many proteins can derive from a single gene (alternative splicing, polymorphism,…).

Each protein can exert several functions in different cellular compartments and its activity is modulate by

environment.

New protein functions can derive from post-translational modifications.

Complexity increase from the genome to the proteome. The number of coding genes is around 20K, but

considering the alternative splicing the number of mRNA is already significantly larger.

The complexity of the proteome is further amplified by tissue-specific expression, PTMs, protein-protein

interactions, and specific subcellular localization. These profiles are dynamically modified upon

biological/pathological perturbations. 1

Dynamic range

Analyzing a cell in different time of its life, its proteome isn’t constant: never the same type and amount of protein.

It’s useful the 2D-PAGE to analyze the different proteomes, in different conditions.

So, a proteome continuously changes and is never the same, increasing the complexity.

How many proteomes in the human body?

A general proteome can be considered, one referring to organs, one referring to tissue and/or one to the cell. Best

is to refer to a sub-proteome to study.

26/02

- Proteomics -

· Definition of proteomics

Proteome analysis is a direct measurement of proteins in terms of their presence and relative abundance (Wilkins

et al., 1996).

Proteomics is the systematic study of the many and diverse properties of proteins in a parallel manner with the

aim of providing detailed descriptions of the structure, function and control of biological system in health and

disease (Patterson & Aebersold, 2003).

· Protein chemistry or proteomics?

Protein chemistry involves the study of protein structure and function. The work generally involves complete

sequence analysis, structure determination and modeling studies to explore how structure governs function.

Proteomics is the study of multiprotein systems in which the focus is on the interplay of multiple, distinct proteins

in their roles.

The starting point is always the tissue, but in protein chemistry the target is just one molecule: isolate the

structure to know the function; while in proteomics the goal is to know how all proteins work together, how they

change in relation with each other. So, global techniques are needed, like 2D-PAGE that gives the general

distribution of all proteins.

· Branches

Proteomics ca be divided in different fields, depending on the application:

- Expression/structural proteomics: all the techniques for protein identification, like MS, and PTMs.

- Quantitative proteomics: to know the difference in the expression levels referring for instance to a control

and/or a pathological state. The difference can be correlated to disease or to a specific function. Must

consider proteins because there’s no way to quantify correctly RNA levels, since RNA is very stable, even if

some techniques are trying. It’s based on 2 types of approach and normally different populations are

tagged with different tags, so the difference in the expression can be evaluated. It’s based on a relative

quantification, not absolute.

- Functional proteomics: to build the network of proteins in the proteome, to understand all the protein-

protein interactions necessary for the life of the cell. A typical technique is the FISH, using a bait to catch

proteins able to interact with one and find the functional relation between proteins.

- Diagnostic proteomics: practical application, the goal is to detect a disease at the early stages of

development and is based on 2 approaches:

Define a pattern, like correlating different expression of a protein to the disease.

o Identify a biomarker and detect it at a very early stage of the disease.

o

- Imaging with MS: to analyze directly tissues or organs.

- Experimental approaches classification -

· Gel or no gel

Separation methods used to simplify the sample, prior the mass spectrometry analysis.

Gel based proteomics

2D gel prior to perform MS. The starting point is the proteome (tissue, organ, leaves, roots, …), obtaining an extract

of soluble proteins, with a technique suited for the cell type.

The mixture is applied on the 2D gel and there’s the separation and it’s possible to compare different images.

Isolate the spots and by an in-situ approach like proteolytic digestion (ex. trypsin), the peptide mixture is formed,

extracted and subjected to MS and MS-MS analysis. Data will be used for the identification of the protein through

research in the database.

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Advantages:

- Mature technology.

- High resolution.

- Identification of PTMs.

- Standard MS capabilities are sufficient.

Limits:

- Low abundant proteins aren’t visible.

- Very hydrophobic proteins can’t be soluble.

- Very small/large proteins: not enter or go out for the gel.

- Very acidic/ basic proteins: at the edge of the gel.

- Labor/time consuming.

Gel free proteomics

The protein mixture is directly analyzed by LC-MS or LC-MS-MS.

The mixture extracted from the tissue is subjected to trypsin digest to obtain a peptide mixture. This is load in the

LC-MS-MS system to obtained set of data to identify all proteins contained in the mixture. So the instrument is

quite advanced and expensive, since it’s a very complex mxture.

Advantages:

- High protein coverage.

- Higher sensitivity.

- Fast, highly automatable, high throughput.

Limits:

- Loss of pI/MW info.

- Loss of peptide-protein connectivity.

- Advance LC/MS/MS required.

- Added complexity.

· Bottom-up or top-down

Refers to the type of analysis of the proteins that are either digested into peptide (bottom-up) or analyzed intact

(top-down).

Bottom-up

Commonly used way, it involves in-gel or in-solution proteolytic digestion of proteins with enzymes, usually

trypsin into small peptides (1-3 kDa), before MS analysis.

This approach is well suited for protein identification, which only requires a small portion of sequence coverage

(10–20 amino acid residues) to identify the protein from the database.

The protein is digested by the enzyme (like trypsin), obtaining peptides and one of them contains the PTM. The

mixture is subjected to MS analysis to identify the MW of each peptide, then the specific m/z (m on z) is isolated

and it undergoes MS-MS to have informations about the structure. Now is possible to obtain the sequence of the

peptide, identify the protein and map modifications.

Shotgun: the bottom-up applied on a mixture of proteins, analogue to shotgun genome sequencing.

Top-down

More sophisticated method. The protein isn’t digested, so it’s intact and loaded in the gas phase of the mass

spectrometer, where it’s fragmentated and the one signal referring to a protein is isolated with an off-set

operation. The protein is disrupted and is subjected to the MS/MS. The spectrum is very crowded since it refers to

the complete protein, with this technique is possible to reveal the sequence of all the protein and the PTMs.

The main advantages include the 100% sequence coverage of the protein and improved detection of post-

translational modifications. 3

· Protein identification

MS based

Using the MW of the peptides, produced by the fragmentation of a protein, to rebuilt the sequence, just comparing

the experimental peptide masses of the spectrometer with the theoretical ones obtained by in silico digestion of all

proteins contained in the genome. There are software able to do that. No information about the structure, just the

MW, only the mass numbers are known → peptide mass fingerprinting.

MS-MS based

It gives MS-MS values, not the mass: data derives from each aa composing the peptide.

This information is used to search in the database and gives informations about the sequence, not the MW.

Always working by comparing analysis data with database data.

- Applications -

Structural proteomics: systematic description of all proteins expressed by a genome.

o Expression proteomics: qualitative and quantitative analysis of a proteome under different conditions.

o Clinical proteomics: identification of protein profile associated to a disease.

o Pharmaceutical proteomics: analysis of the effects of drugs or medicines on gene expression.

o Functional proteomics: study of protein complexes and protein-protein interaction.

o Quantitative proteomics: determination of the relative abundance of the protein expression level.

o

01/03 Peptide Mass Fingerprinting

It’s an operation of rebuilding of the sequence of a molecule upon fragmentation in small pieces.

It’s independent from the chosen experimental approach.

- Fragmentation -

· Chemical fragmentation

Advantages: specificity and infrequency of the susceptible peptide bonds.

Suitable for the generation of relatively simple mixtures of large fragments.

Limit: side reactions that produce new species that increase the complexity of the mixture, the modify protein

doesn’t match to the original one.

Chemical reagents and cleavage sites:

Cyanogen bromide: C-terminus of M (M-X bond).

§ Partial acid hydrolysis (HCl, formic acid, acetic acid): aspartyl residues (D-P, D-G).

§ Hydroxylamine: N-G bonds.

§ BNPS-skatole: W residues.

§

· Enzymatic fragmentation

Proteolysis: specific hydrolysis of one or more peptide bonds by an enzyme. Mechanism: S + E ⇔ ES ⇔ E + P

Limited proteolysis: rigorous controlled conditions as pH temperature., time, to cleave the protein only on a limited

number of peptide bonds. Usually this proteolysis gives structural informations.

Exhaustive proteolysis: very harsh conditions and gives many and small peptides, cleaving as much as possible to

obtain the primary structure of the protein.

Roles in biology:

• Zymogens activation.

• Hormone production from protein precursors.

• Intracellular protein turnover.

• Post-translational processing of proteins.

• Transport across membranes.

• Blood coagulation.

• Apoptosis.

• Many enzymes are activated by specific cleavage.

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Useful to:

Analyze protein structure.

o Identify disordered regions within folded proteins.

o Follow dynamic processes and the evolution of protein conformation.

o

Mechanism:

1. Proteins are treated with a proteolytic enzyme.

2. Peptides can be isolated.

3. Peptides are identified.

4. The primary structure can be rebuilt

Nomenclature:

To describe the interaction of a polypeptide substrate at the protease’s active site.

The residues of the substrate bind at subsites of the protease’s active site, they’re called S and the substrate

residues are called P.

The residues of the N-terminal side of the scissile bond are numbered P3, P2, P1 and those residues of the C-

terminal side are numbered P1', P2', P3'... The P1 or P1' residues are those residues located near the scissile bond.

Sometimes proteases doesn’t recognize P1, but it does before and cleave at P1.

In several studies it has been established that the protease-peptide/protein substrate interaction involves a

stretch of up to 12 residues, so it’s important to define he position of them.

- Experimental procedure of proteolysis -

· Choice of the protease

Exopeptidase: cut at the terminal of the peptide, first or last aa. Exo. recognizing dipeptide

Endopeptidase: cut in the middle of the sequence, useful for proteomics approach.

There are many possible enzymes, they can be bought.

Protease MM pH Specificity Inhibitors Notes

(kDa)

Trypsin 23.5 8-9 Arg, Lys PMSF, DFP, TLCK, Due to its narrow specificity, it

aprotinin, leupeptin, can cut at disordered regions of

soybean trypsin proteins only if these contain Lys

inhibitor. or Arg residues. It does not cleave

the Lys-Pro or Arg-Pro peptide

bond. If an acidic residue is on

either side of the cleavage site, the

rate of hydrolysis is much

reduced. Trypsin preparations

may contain traces of “pseudo-

trypsin” that cleaves at

hydrophobic amino acid residues

as α-chymotrypsin.

Other features: high frequency of

R and K, so short and many

peptides, high sequence coverage;

tryptic peptide with good MS-MS

signal (2 positive charges).

Subtilisin 27.3 7-11 Non-specific α -macroglobulin, It’s the most useful for detecting

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PMSF, DFP. sites or regions of protein

disorder.

Thermolysin 34.9 7-9 Leu, Ile, Phe, 1,10-phenathroline, It cuts at the N- terminus of

Val, Met, Ala α -macroglobulin, mostly hydrophobic residues. For

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phosphoramidon, optimal stability it may be used in

EDTA, citrate, the presence of 1-10 mM CaCl

2

phosphate. and, being a thermostable

enzyme, at rather high

temperature (up to 80°C). 5

α-chymotrypsin 25 7-9 Phe, Tyr, Trp, DFP, PMSF, TPCK, Moderate specificity for aromatic

Leu, Ile. soybean trypsin and hydrophobic amino acid

inhibitor, chymostatin, residues, useful for detecting

heavy metals. disordered sites in proteins.

Proteinase K 28.9 7.5- Non-specific DFP, PMSF, Hg It usually cuts at several residues

++

11 within a disordered region of a

protein; it is not inactivated by

EDTA, sulfhydryl reagents and

TPCK.

· Choice of the experimental conditions

Specificity

The specificity of a protease is never absolute, but depends on the interaction between the proximal residues of

the peptide bond to be cleaved and the protease binding site.

- Primary specificity: is defined by the side chain in position P1 or P1’ that adapts to S1.

- Secondary specificity: is present in some enzymes, they are important for the interaction with the substrate,

even if the residues are localized in position far from the peptide bonds to be hydrolyzed.

pH/temperature optimum

Different proteases has different pH optimum: pepsin works best at acidic pH while trypsin in more neutral/basic

pHs. Same thing for temperature.

Changing the pH to stop the activity it’s not a good idea if it’s not completely clear which is the range of activity of

the enzyme, same thing for the temperature.

Stability to denaturing agents

• Denaturing agents (urea, guanidine).

• Organic solvents (alcohol, DMSO).

• Detergents (SDS).

• Lipids (fatty acids, phospholipids).

Be careful: Chymotrypsin specificity changes in water and octane

Native/denatured state

A protein in the native state has accessible only peptide bonds not involved in secondary structure, while in the

denatured state there’s just the linear polypeptide chain and the complete cleavage is possible.

Role of S-S bridges

Disulfide bridges reduction makes peptide bonds more accessible to proteases and so the proteolytic reaction is

more efficient. Reducing agents: DTT, TCEP, β-mercaptoethanol.

Iodoacetamide can be used to block the formation of S-S, since it bind covalently the thiol group of Cys.

· Controlling the proteolytic digestion

E:S ratio should be specified, because proteolysis is a bimolecular reaction dependent on the concentrations of

both the proteolytic enzyme and the protein substrate.

E:S ratios commonly used are 1:100, but 1:1000 or even 1:10,000 are sometimes used.

Possible ways to control proteolysis is by using a low concentration of protease, short reaction times and low

temperature.

· Inhibition/inactivation of the proteolysis

Change of the pH solution (where enzyme’s activity is inhibited).

§ Freezing, knowing the. T range of activity of the protease.

§ Adding chelating agent for metal-proteases.

§ Adding of specific inhibitors for proteases.

§

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04/03

· Isolation of peptides

How to resolve the mixture: electrophoresis (SDS-PAGE) and chromatographic methods (RP-HPLC, IEX-HPLC).

SDS-PAGE: it’s relatively cheap,