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The quantification of proteins is essential for studying cellular processes and functions. It serves as a powerful tool in understanding the basic biological processes and mechanisms. This essay highlights the various methods that are currently available for relative quantification of individual proteins in complex biological molecules using the technique of mass spectrometry. The field of quantitative mass spectrometry is under fast development with new and better instrumentation being developed every year. However there is no single method that is considered superioras all have theirrespectivefield of application, each having its own merits and drawbacks.
METHODS AND APPROACH
There are basically two different methods for quantifying proteins using mass spectrometryviz. "Bottoms up" approach - where the protein is digested into peptides with the help of protease followed by its quantification through spectral information by mass spectrometry and "Top-down" approach - where the whole protein is separated and analysed individually by a mass spectrometer.
There are three different approaches to quantitative protein profiling. ,
Proteins are initially separated and quantified by 2D-GE and then identified by mass spectrometer.
Protein expression array analysis - It involves the generation of ordered arrays of reagents with high specificity for particular proteins. It is based on the linear translation of mRNA expression array concept into proteomics.It is the one that is least developed
Stable isotope labelling of proteins and automated tandem MS/MS. Here the different peptides are labelled with a stable isotope. As a result they differ in their mass and also exhibit some chemical properties during chromatographyand are then compared and quantified. The label can be introduced at various steps during sample preparation (SeeTable: 1).
Metabolic Labelling Techniques
In this method, the label introduced to the whole cell/organism through the growth medium.
SILAC - Stable Isotope Labelling in Cell Cultures
It provides a high throughput quantitative protein analysis based on metabolic labelling. It is an in vivo labelling of proteins in cell culture with amino acids that contain stable (non-radioactive) isotopes like C13 and N15 . The cell culture pair is grown under identical conditions except that one of the cultures is given an isotopically labelled amino acid. Labelled (Heavy) and Unlabelled (light) fragmented peptide isoforms is subjected to extracted ion chromatography (See Fig: 1). As the physicochemical properties of the peptides do not change due to the isotope label, the heavy and light forms usually co-elute off the chromatographic gradient.
Depending on the operating parameters of the mass spectrometer, each survey scan can be done every 1-5 seconds and this allows the sampling of several MS full scan spectra. Each spectrum is a single observation of the peptide pair and the average of the ratio will give the standard deviation for each peptide. The abundance ratio between the heavy and light forms is can be done by two methods. It can be done at each of the survey scan after which the individual ratios are averaged. Thus each ion will have an average ratio and a standard deviation or alternately the ratio can be calculated between the peak volumes and the total extracted ion chromatogram (This method is less affected by slight shifts in chromatographic elution).
It works well in bacterial or specific yeast strains which is auxotrophic to the labelled amino acid.
Inexpensive non-radioactive method for labelling protein.
Can be used invitro and in vivo.
In complex samples there can be signal interferences caused by co-eluting components of similar mass.
It can be used only for the analysis of organisms whose growth media can be controlled
It can lead to higher ambiguities as the large number of incorporated labelled atoms in the peptide sequence can generate more isobaric amino acids forms.
SILT - Stable Isotope Labelling Technique
It is a method to quantify production and clearance rates for low abundance proteins using tandem mass spectrometry. . In this technique, the protein is labelled by the incorporation of stable isotope labelled amino acids. Since a very small concentration of the protein is present, it is isolated and concentrated by methods like immunoprecipitation. This is followed by the proteolytic cleavage of the protein into smaller peptides and the amounts of labelled and unlabelled tandem MS ions are calculated.
1. Can label proteins in vitro and in vivo.
2. Can be quantified even when present in very low amounts.
3.Non-radioactive method for labelling proteins.
4. All proteins are labelled simultaneously.
5. The technique is reproducible and can be adapted to simultaneously quantify several proteins degraded invitro/invivo systems.
1.Care must be taken while selecting the appropriate stable isotope labelled precursor. It must not rapidly enter recycling metabolic processes.
2. When protein samples like amyloid-Î² (for Alzheimer's disease) is analysed, the stable isotope selected should cross the blood brain barrier.
Chemical Labelling techniques
In this method the label is introduced to the peptides by a chemical reactionthrough sulfhydryl group or amine group, or through esterification or acetylation of amino acid residues . Here the quantitation is based on full scans and the mass shift between the heavy and light peptides is constant. However for clear quantification, the mass difference between the heavy and light forms must be at least 4 Da and hence cannot be used for very large peptides.
ICAT - Isotope Coded Affinity Tag
The ICAT reagent consists of a reactive group that is binds to the sulfhydryl group of cysteine residues. The isolated proteins are denatured, reduced and modified with the ICAT reagent. The use of isotope produces a pair of chemically identical reagents with the heavy reagent weighing slightly more than the other. The ICAT labelled peptides are then eluted and quantified with MS. However cysteine beinga less abundant amino acid,the number of peptides that can be tagged and quantified by sulfhydryl groups is low.
ISOBARIC MASS TAG
In this methods, the addition of the mass tags leads to the generation of labelled peptides that have the same total mass. As a result they co-elute and produce only a single peak in liquid chromatography. The mass tags can be distinguished only after peptide fragmentation. TMT and iTRAQ are commercially available isobaric mass tags that are introduced to the peptides.
TMT - Tandem mass tag
These tags are designed in a manner that on analysis by CID - Collision Induced Dissociation, the TMT fragment (comprising of sensitization group and mass differentiation group) is released to give ion with specific charge to mass ratio . It is a more precise technique than isotope labelling method as the TMT fragment ions accurately reflect the relative abundance of the peptides from which they are derived. Also the tags show consistent property over a wide range and since the tagged peptides co-elute, the ratios of the peptide pairs are conserved.
iTRAQ - Isobaric Tag for Relative and Absolute Quantitation.
It is an in vitro chemical labelling method.iTRAQ reagent consists of four isobaric amine specific tags. It contains 3 regions (See Fig. 2)viz. a peptide reactive region, a balance region and a reporter region (Ross et al., 2004). The protein is individually reduced to break the disulphide bonds, alkylated to block free sulfhydryls or cysteines and then digested with a peptide to create peptides.Each sample is then separately labelled with one of the four iTRAQ reagents.The four samples are then combined into one mixture which is then analysed by mass spectrometry. The labelled peptides produce fragmentation spectra that help in the identification of the protein.Protein quantification is achieved by comparing the intensities of the four reporter ion in the MS/MS spectra with the help of software like ProQuant and iTracker
1. Four samples can be identified simultaneously
2. It gives higher MS/MS intensity and therefore gives better peptide identification as compared to ICAT, SILAC and metabolic labelling.
3. It can be used to quantify clinical samples like tumour tissues as it is an in vitro labelling technique.
Mudpit - Multi Dimensional Protein Identification Technology
In this method the proteome is digested and separated using dual phase liquid chromatography (consisting of strong cationic exchanger and reverse phase matrix material) and ESI. No additional sampling is required as the peptides, after eluting from the column are directed into the ESI ion trap mass spectrometer. Advanced search algorithms like SEQUEST was used to match the fragmented peptides to the respective proteins in the database . It has the advantage of giving a complete representation of proteins; however it leads to high redundancy and sample complexity.
LABEL FREE APPROACHES
It compares two or more samples in parallel based on the ion intensity of identical peptides or based on the number of acquired spectra for each protein . It is inexpensive and can be applied to any biological material and the proteome coverage of quantified protein is high as every protein that is identified by the spectra can be quantified. However it gives relatively poor precision.
CONCLUSION AND DISCUSSION
Quantitative proteomics is a relatively new and challenging field taking into consideration the complexity and dynamic nature of the proteome. Relative quantification method as described in this essay work on the principle of comparing the value of one protein with another protein. The various methods described here are not absolute and have their merits and drawbacks. These techniques help in the better understanding of the various biological processes; however there are cases where the analysis was difficult to interpret. Hence the performance of the techniques needs to be improved further through better instrumentation and more efficient algorithm and software, so as to overcome the drawbacks.
Table: 1 - Different types of labelling
Label introduced to the whole cell/organism through the growth medium.
Label added through chemical reaction. E.g. Sulfhydryl group or amine group or through esterification or acetylation of amino acid residues.
Standard peptides are added to the extract after tryptic digest.
Fig: 1: SILAC Technique.
Fig: 2 - iTRAQ Technique