Also, there are antibodies specific to other modifications, which can be used to determine the set of proteins that have undergone the modification of interest.

Development of antibody specific to that modification can be a way out to study a particular protein. Therefore, no single technique including high throughput can provide acceptable answer to possible modifications. Besides differences caused due to translation from mRNA, many proteins undergo wide variety of chemical modifications after translation. Previous approach of mRNA analysis was found not to correlate with protein content as amount of protein produced for a given amount of mRNA depends on the gene it is transcribed from and on the physiological state of the cell. Proteome differs from cell to cell and from time to time per se distinct genes are expressed in different cell types making it essential to identify even the basic set of proteins that are produced in a cell.

Post-translational modifications are critical to the protein's function. Several tools are most commonly used to perform this task, although the most used are Mascot (Matrixscience), Sequest, Maxquant and Paragon. Unless targeted approaches are used, the characterization of PTMs using MS/MS data involves exhaustive searches of all potential combinations of mass shift for each identified peptide from a protein database. Of all the work-flow tasks, data processing, analysis and presentation are probably the most challenging aspects of proteomics. High-throughput proteomics allows the simultaneous identification of thousands of modified peptides. These are essentially the same as those used for protein identification. There are several strategies for PTM identification using mass spectrometry. Currently, several approaches are used for identifying and quantifying proteins and PTMs, which have been conveniently reviewed in several high quality papers. To understand the complexity of the problem, Thelen and Miernyka reported a virtual comparison of a liquid chromatography profile of a peptide containing zero to three PTMs, which highlighted the need to use a combination of strategies to improve the identification of PTMs. Also it is difficult, if not impossible, to devise a strategy to allow the separation and analysis of all structurally diverse protein isoforms, because of the number of possible combinations of PTM locations within a protein with several PTMs. As mentioned in the previous section, the very high dynamic range of protein concentrations in a cell poses serious difficulties for the identification of PTMs. high purity with low degradation and low abundance of contaminants, by the use of competitive separation techniques for intact proteins or peptides. However, for the unequivocal assignment of a given modification site, tandem mass spectrometry (MS/MS) experiments are necessary to show that the mass shift detected in the precursor ion (the peptide from tryptic digestion) is also observed in the fragment ions carrying the modified amino acid residue.Īn essential aspect of PTM identification is the need for high quality samples, i.e. Detection of PTMs by mass spectrometry relies on alteration of the mass of tryptic peptides, which results from chemical modification occurring in the amino acid residue side chain.

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