Proteomics: Techniques and Applications in Biological Research | Study notes Genomics

GAP- UNIT-5

The word “proteome” (term coined by Mark Wilkins in 1995) represents the complete

protein pool of an organism encoded by the genome. In broader term, Proteomics, is

defined as the total protein content of a cell or that of an organism. Proteomics helps in

understanding of alteration in protein expression during different stages of life cycle or

under stress condition. Likewise, Proteomics helps in understanding the structure and

function of different proteins as well as protein-protein interactions of an organism. A

minor defect in either protein structure, its function or alternation in expression pattern

can be easily detected using proteomics studies. The first protein studies that can be

called proteomics began with the introduction of two-dimensional gel electrophoresis of

E. coli proteins (O’Ferrall, 1975) followed by mouse and guinea pig protein studies (Ksole,

1975). This is important with regards to drug development and understanding various

biological processes, as proteins are the most favourable targets for various drugs.

Proteomics on the whole can be divided into three kinds.

Structural Proteomics:

One of the main targets of proteomics investigation is to map the structure of protein

complexes or the proteins present in a specific cellular organelle known as cell map or

structural proteins. Structural proteomics attempt to identify all the proteins within a

protein complex and characterization all protein-protein interactions. Isolation of

specific protein complex by purification can simplify the proteomic analysis.

Functional Proteomics:

It mainly includes isolation of protein complexes or the use of protein ligands to isolate

specific types of proteins. It allows selected groups of proteins to be studied its

chracteristics which can provide important information about protein signalling and

disease mechanism etc.

Differential Proteomics:

This includes determination of difference in protein expression.

Techniques Involved in Proteomics Study

Some of the very basic analytical techniques are used as major proteomic tools for

studying the proteome of an organism. The initial step in all proteomic studies is the

separation of a mixture of proteins. This can be carried out using Two-Dimensional Gel

Electrophoresis technique in which proteins are first of all separated based on their

individual charges in 1D. The gel is then turned 90 degrees from its initial position to

separate proteins based on the difference in their size. This separation occurs in 2nd

dimension hence the name 2D. The spots obtained in 2D electrophoresis are excised and

further subjected to mass spectrometric analysis of each protein present in the mixture.

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The word “proteome” (term coined by Mark Wilkins in 1995) represents the complete protein pool of an organism encoded by the genome. In broader term, Proteomics, is defined as the total protein content of a cell or that of an organism. Proteomics helps in understanding of alteration in protein expression during different stages of life cycle or under stress condition. Likewise, Proteomics helps in understanding the structure and function of different proteins as well as protein-protein interactions of an organism. A minor defect in either protein structure, its function or alternation in expression pattern can be easily detected using proteomics studies. The first protein studies that can be called proteomics began with the introduction of two-dimensional gel electrophoresis of E. coli proteins (O’Ferrall, 1975) followed by mouse and guinea pig protein studies (Ksole, 1975). This is important with regards to drug development and understanding various biological processes, as proteins are the most favourable targets for various drugs. Proteomics on the whole can be divided into three kinds. Structural Proteomics: One of the main targets of proteomics investigation is to map the structure of protein complexes or the proteins present in a specific cellular organelle known as cell map or structural proteins. Structural proteomics attempt to identify all the proteins within a protein complex and characterization all protein-protein interactions. Isolation of specific protein complex by purification can simplify the proteomic analysis. Functional Proteomics: It mainly includes isolation of protein complexes or the use of protein ligands to isolate specific types of proteins. It allows selected groups of proteins to be studied its chracteristics which can provide important information about protein signalling and disease mechanism etc. Differential Proteomics: This includes determination of difference in protein expression. Techniques Involved in Proteomics Study Some of the very basic analytical techniques are used as major proteomic tools for studying the proteome of an organism. The initial step in all proteomic studies is the separation of a mixture of proteins. This can be carried out using Two-Dimensional Gel Electrophoresis technique in which proteins are first of all separated based on their individual charges in 1D. The gel is then turned 90 degrees from its initial position to separate proteins based on the difference in their size. This separation occurs in 2nd dimension hence the name 2D. The spots obtained in 2D electrophoresis are excised and further subjected to mass spectrometric analysis of each protein present in the mixture.

Steps in Proteomic Analysis The following steps are involved in analysis of proteome of an organism as :

Purification of proteins: This step involves extraction of protein samples from whole cell, tissue or sub cellular organelles followed by purification using density gradient centrifugation, chromatographic techniques (exclusion, affinity etc.)
Separation of proteins: 2D gel electrophoresis is applied for separation of proteins on the basis of their isoelectric points in one dimension and molecular weight on the other. Spots are detected using fluorescent dyes or radioactive probes.
Identification of proteins: The separated protein spots on gel are excised and digested in gel by a protease (e.g. trypsin). The eluted peptides are identified using mass spectrometry.

Ampholytes of a given range (for example pH 2.0-12.0) is mixed with acrylamide/bis acrylamide while being polymerized (no SDS). After electrophoresis is performed the ampholyte molecule are arranged according to their isoelectric point and create a pH gradient. As ampholytes have high buffering capacity at isoelectric point a higher pH is maintained. Working Principle

If we do an electrophoresis in pH gradient and protein sample (protein mixture) is loaded at pH 7.0:
All proteins with isoelectric point above pH 7.0 will have positive charge and move towards negative electrode.
As they move towards negative electrode due to pH increase (due to pH gradient) net positive charge on protein would decrease.
When protein reaches pH which equals isolectric point of protein, net charge on the protein becomes zero and electrophoretic mobility becomes nil (protein remains confined near isoelectric point in the pH gradient.
All proteins with isoelectric point below 7.0 will have negative charge and move towards positive electrode.
As they move towards positive electrode there is a decrease in the net negative charge on the protein due to pH decrease (due to pH gradient).
When protein reaches pH which equals isolectric point of protein, net charge on the protein becomes zero and electrophoretic mobility becomes nil.

After first dimension run, strip gel is placed on the top of SDS-PAGE gel horizontally. The protein is already denatured in first dimension (by urea and reducing agents). Thus, the SDS used in gel running buffers sufficient to bind with already denatured protein maintain the necessary uniform negative charge for SDS-PAGE. Once the second dimension run is over, gel is separated and stained for protein visualization using methods studied in SDS-PAGE (Coomassie blue staining, Silver staining etc.)

Polysaccharides Polysaccharides can interfere with electrophoresis by clogging gel pores and by forming complexes with proteins. Centrifugation may be used to remove high molecular weight polysaccharides. Phenol extraction, followed by precipitation with ammonium acetate in methanol, is a commonly used method that is very effective at removing polysaccharides in plant samples. Phenolic Compounds Phenolic compounds are found in all plants and in some microorganisms and they can modify proteins. Phenolic compounds may also be removed from the extraction by including polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP) in the extraction solution. These compounds bind phenolic compounds, and the precipitated complex can be removed from the extract by centrifugation. Lipids Lipids can form insoluble complexes with proteins, but lipids can also complex with detergents, thereby reducing the detergents’ effectiveness at solublilizing protein. By adding organic solvent like phenols and chloroform lipids are separated from the sample after centrifugation. Proteases Cell disruption methods destroy the compartmentalization of a cell, causing the release of hydrolases (phosphatases, glycosidases, and proteases). These enzymes modify proteins in the lysate. So protease inhibitors are added to the lysis solution. Examples include either small molecules, such as phenylmethylsulfonyl fluoride (PMSF), aminoethyl-benzene sulphonyl fluoride (AEBSF). Solubilization of protein: Proteins in a biological sample are often associated with other proteins, integrated into membranes, or parts of large complexes. Protein solubilization is the process of breaking interactions involved in protein aggregation. which include disulfide and hydrogen bonds, van der Waals forces, and ionic and hydrophobic interactions. If these interactions are not disrupted, proteins can aggregate or precipitate, resulting in artifacts or sample loss. For successful 2 - D electrophoresis, proteins must be well solubilized. Chaotropic Agents These compounds disrupt hydrogen bonds and hydrophobic interactions both between and within proteins. When used at high concentrations, chaotropic agents disrupt secondary protein structure. The neutral chaotropic agent urea is used at 5–9 M, often with up to 2 M thiourea, which can dramatically increase the number of proteins solubilized.

Detergent Solubilizes the proteins and helps to maintain the proteins in solution during IEF. Total detergent concentration range is 0.5–4%. Sample preparation for 2-D electrophoresis commonly uses neutral or zwitterionic (having both positive and negative charges resulting in a neutral net charge) detergents at concentrations of 1–4%, since these detergents do not introduce a net charge and therefore allow proteins to migrate at their own charges during IEF. Examples of neutral detergents include Tween, octylglucoside, dodecyl maltoside, Triton X-100, and Triton X- 114. Reducing agent Reducing agents cleave disulfide bond cross- links within and between protein subunits, thereby promoting protein unfolding and maintaining proteins in their fully reduced states. The compounds used for 2-D sample preparation are dithiothreitol (DTT), dithioerythritol (DTE), and β-mercaptoethanol (BME).

Working principle of Mass Spectrometry.

Sample inlet : In a typical procedure, a sample, which may be solid, liquid, or gas, is convert to gaseous phase in the sample inlet chamber by the heating coil.
Ionization: Then the gaseous sample enters to the ionization chamber. A beam of electron bombards with the molecule and remove one electron from it. Now the sample M converts to M+ ion.
This also may cause some of the sample’s molecules to break into charged fragments. These ions are then separated according to their mass-to-charge ratio after acceleration phase.
There are several types of ionization methods in mass spectrometry. The physical basis of ionization methods are very complex and outside the scope of the course. Most common methods are:
Electron impact ionisation (EI): Electron impact ionisation (EI) is widely used for the analysis of metabolites, pollutants and pharmaceutical compounds, for

example in drug testing programmes. A stream of electrons from a heated metal filament is accelerated to 70 eV potential .Interaction with the analyte results in either loss of an electron from the substance (to produce a cation).

Matrix-assisted laser desorption/ionization (MALDI)
This method of ionization is a soft ionization method and results in minimum fragmentation of sample. This method is used for non-volatile, and thermally labile compounds such as proteins, oligonucleotides, synthetic polymers. Sample spotted onto a metal plate and dried. Matrix plays a key role in this technique by absorbing the laser light energy and causing a small part of the target substrate to vaporize. Although, the process of forming analyte ions is unclear, it is believed that matrix which has labile protons, such as carboxylic acids, protonates neutral analyte molecules after absorbing laser light energy.
In the acceleration chamber ,Ions are accelerated so that they all have the same kinetic energy and they can move towards the mass analyser. Positive ions pass through 3 different electrodes attached to the wall. Negative electrode will accelerate the M+ ions.
In the deflection phase the ions enters to the magnetic field. Ions are deflected by a magnetic field due to differences in their masses.
The lighter the mass, the more they are deflected. It also depends upon the no. of +ve charge an ion is carrying; the more +ve charge, the more it will be deflected.
Detection: The beam of ions passing through the mass analyzer is detected by a detector on the basis of the m/e ratio. When an ion hits the metal box, the charge is neutralized by an electron jumping from the metal onto the ion.

De novo sequencing De novo is Latin which means “over again” or “a new”. The de novo peptide sequencing is a method for peptide sequencing performed without prior knowledge of the amino acid sequence. This method can obtain the peptide sequences without a protein database, which can overcome the limitations of database-dependent methods like peptide mass fingerprinting (PMF). It uses computational approaches to deduce the sequence of peptide directly from the experimental MS/MS spectra. It can be used for un-sequenced organisms, antibodies, peptides with posttranslational modifications (PTMs), and endogenous peptides. Method:

In this method, the peptide is fragmented along the peptide backbone and the resulting fragment ions are measured to produce spectra.
There are 3 ways to break bonds to form peptide fragment: alkyl carbonyl (CHR- CO), peptide amide bond (CO-NH), and amino alkyl bond (NH-CHR).
Therefore, it can form 6 types of fragmentation ions, including the N-terminal charged fragment ions which are classed as a, b, or c, and the C-terminal charged ones which are classed as x, y, or z.
And because the peptide amide bone (CO-NH) is the most vulnerable, the most common peptide fragments observed in low energy collisions are a, b and y ions.
De novo methods use the knowledge of the fragmentation methods employed in the (Mass spectrometry) MS. CID, Collision induced dissociation, also known as collisionally activated dissociation, is the most common form of fragmentation of the molecules into smaller fragments. This method results in the formation of b and y series ions from the precursor ion. The Electron capture dissociation (ECD) and Electron transfer dissociation (ETD) have been implemented in the recent mass spectrometer. In these methods, Ions are fragmented after reaction with electrons. After fragmentation, it forms c and z type ions through cleavage of the peptide bond between the amino group and alpha carbon. The mass can usually uniquely determine the residue.

The main principle of de novo sequencing is to use the mass difference between two fragment ions to calculate the mass of an amino acid residue on the peptide backbone.
If one can identify either the y-ion or b-ion series in the spectrum, the peptide sequence can be determined. However, the spectrum obtained from the mass spectrometry instrument does not tell the ion types of the peaks, which need an expert or a computer algorithm to figure out.
There are couples of software packages used for de novo sequencing, such as PEAKS, Lutefisk, PepNovo, SHERENGA, etc.

Proteomics: Techniques and Applications in Biological Research, Study notes of Genomics

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