Protein Identification Strategies

Clauser, K. R. Baker, P Burlingame, A. L. Role of accurate mass measurement (+10ppm) in protein identification strategies employing MS or MS/MS and database searching. Anal. Chem. 1999,71,2871-2882. 

Protein Identification Strategies 15.2.8.1 Peptide Mass Fingerprinting (PMF)... 

Clauser, K.R. Baker, P. Burlingame, A. Role of Accurate Mass Measurement ( 10 Ppm) in Protein Identification Strategies Employing MS or MS/MS and Database Searching. Anal. Chem. 1999, 71, 2871-2882. 

Figure 4.2 Schematic overview of two protein identification strategies commonly followed in proteomics. Protein samples are separated by either two-dimensional (2-D) or one-dimensional (1 -D) polyacrylamide gel electrophoresis (PAGE). In both strategic tracks, proteins are converted into a set of peptides by enzymatic digestion (e.g., with trypsin) prior to MS analysis. Peptide mass fingerprinting (PMF) by MALDl MS is predomi-...

The bottom-up approach very much resembles classical protein identification strategies. The proteins in the proteome are first separated by 2D-GE (Ch. 17.3), or in some cases by SCX, size-exclusion (SEC), or affinity (AfC) chromatography. Specific proteins are excised from the gel, blotted, or electroeluted. The protein is digested, and the digest is analysed by LC-MS. The EC separation involves either RPLC with microcapillary or nano-LC columns (Ch. 17.5.2), or 2D-LC with typically SEC or SCX in the first dimension and RPLC in the second (Ch. 17.5.4). Alternatively, the sample may be introduced via either direct-infusion nano-ESl (Ch. 17.2), CE-MS (Ch. 17.5.6), or a microfluidic device coupled to MS (Ch. 17.5.5). 

While all these strategies rely on specific properties of phosphopeptides in MS analysis, a more global approach involving shotgun protein identification strategies and SEQUEST database searching (Ch. 18.3.2) is demonstrated by the group of Yates for protein complexes and lens tissue proteins [29]. 

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