Proteomics workflow

Figure 10.5. Proteomic workflow via protein separation, digestion, MS analysis, and database search.

Before examining contemporary proteomic workflows, it must be said that the older gel-based technology still remains the largest contributor of proteomic-level information. For many years, the main means to analyze complex protein samples involves separation of proteins on the basis of size and isoelectric point (pi) using two-dimensional polyacrylamide gel electrophoresis (2-DE). The densitometric aspects of gel protein spots provide a direct means to visualize and quantify proteins based on the experimental conditions applied to the... 

One clear aspect of the current trends in the proteomic field is the plethora of ways to achieve informational outcomes. Research developments over recent years have focused on all facets of the proteomic workflow including improvements in the comprehensiveness of analysis, efficiency of workflows, and development of specialized methods. This is aided by several mass spectrometers being available on the market that have the necessary accuracy, sensitivity, speed, and dynamic ranges. The baseline workflow for proteomics however remains essentially the same and involves a number of required steps (summarized in the succeeding text and in Figure 1) ... 

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. 

Holman, S. W., Sims, P. F. G., and Eyers, C. E. 2012. The use of selected reaction monitoring in quantitative proteomics. Bioanalysis 4 1763-1786. Picotti, P., and Aebersold, R. 2012. Selected reaction monitoring-based proteomics workflows, potential, pitfalls and future directions. Nat. Methods 6 555-566. 

Picotti P, Aebersold R. Selected reaction monitoring-based proteomics workflows, potential, pit-falls and future directions. Nat Methods. 2012 9(6) 555-66. 

Gundry RL, White MY, Murray Cl, et al. Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Curr Protoc Mol Biol. 2009 88 10.25.1-10.25.23. doi 10.1002/0471142727.mbl025s88. 

Pierce CL, Rees JC, Fernandez FM, Barr JR. Viable Staphylococcus aureus quantitation using N-15 metabolically labeled bacteriophage amplification coupled with a multiple reaction monitoring proteomic workflow. Mol Cell Proteomics. 2012 11 M111.012849. doi 10.1074/mcp. Mlll.012849-5. 

Figure 4.7 In-lysate chemical proteomics workflow, (a) Sample preparation from disease-relevant cellular lysate or tissue (b) competition with investigational compound active and inactive of the identical chemotype ...

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