Proteomics analysis process

The combination of this top-down proteomics approach, which generates information on the structure of the intact protein, with a bottom-up approach for protein identification (using MS/MS data of tryptic peptides from the collected fractions) has been particularly useful for identifying posttranslational modifications, cotransla-tional processing, and proteolytic modifications in a number of proteins. Examples from our work will be shown to illustrate this hybrid methodology for proteomics analysis. 

The subsequent downstream processing section, which includes visualization of quantified proteins, statistical validation of differences in treatments or samples, and biological interpretation, is much less defined in terms of work-flow regimens and is discussed toward the end of this chapter. In the succeeding text, various relevant aspects of proteomic workflows that impinge on the data obtained from proteomic analysis of prokaryotes assuming that a gel-free approach is used are discussed. 

A proteomic analysis of a sample usually consists of four steps. These are extraction of the proteins from the sample, their separation, detection, and finally identification/analysis of the individual separated proteins. Major attention must be paid to the sample processing, sample handling, and the sample clean-up since any error or sample loss during this stage influences the final result. 

The domain of applications in proteomic analysis are abundant and diverse. This method can be used to understand important biological processes involved in cell... 

One of the most crucial considerations in proteomic analysis is sample preparation because this will ultimately dictate the number and type of proteins that can be processed. The first priority is to establish the precise protein system to be studied [e.g., will this be a comprehensive and exhaustive catalogue of every expressed protein within a tissue or cellular extract, or is only a small subset of a cellular proteome (e.g., only phosphoproteins or membrane-bound proteins) sufficient for analysis ]. Whether a full or partial proteome, or even a limited number of specific proteins is required for analysis, it is crucial that the extraction technique provide maximal protein recovery while preserving the integrity of the protein complex to be examined. Furthermore, the method of preparation must be totally compatible with the separation methods to be used. This is particularly important for separation technologies that are reliant on protein-protein interactions or drug/ligand/antibody, etc. interactions. Poor recovery of proteins is clearly... 

Similarly, we have described how information related to protein-splicing variants, amino acid peptide variants (polymorphisms), and PTMs is being introduced in data analysis pipelines in order to increase the rate of identifiable peptides. The introduction of genomics and transcriptomics information, often neglected in routine proteomics analyses, will enable the characterization of myriad splicing variants and amino acid polymorphisms, as well as the development of robust proteomics analysis pipelines, which will contribute to elucidate their role in health and disease states. These tools will make it possible to uncover a new layer of the proteome complexity and to expand the information available from cellular systems and the potential implication of these protein variants in different physiological processes. Finally, several bioinformatics tools that perform unbiased PTM analysis have also been reviewed. The tools that are currently being developed and refined tackle... 

Since light and heavy amino acids are chemically identical, the labeling process will not affect the chemical properties of the peptides and therefore differentially labeled peptides will co-elute from the HPLC column. However, these peptides are isotopically distinct from each other the peaks from light and heavy labeled peptides can be accurately distinguished and quantified by using mass spectrometry. An example of a study using SILAC includes the quantitative proteomic analysis of 495 proteins in renal cells towards the exploration of molecular mechanisms of calcineurin-inhibitors induced nephrotoxicity [82], In a second study, a... 

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