Proteome coverage peptides

L. Pasa-Tolic R. Harkewicz, G.A. Anderson, M. Tolic, Y. Shen, R. Zhao, B. Thrall, C. Masselon, R.D. Smith, Increased proteome coverage for quantitative peptide abundance measurements based upon high performance separations and DREAMS FT-ICRMS, J. Am. Soc. Mass Spectrom., 13 (2002) 954. 

No current analytical strategy is capable of fully resolving complex biological samples. For this reason, orthogonal separation techniques are often combined to maximize peptide separation before the mass spectrometric analysis. The aim is to reduce as much as possible the number of coeluting peptides introduced into the MS at any given time, so as to maximize peptide identification and proteome coverage. 

Other proteolytic enzymes commonly used in proteomics analysis include Lys-C, Lys-N, and chymotrypsin. In general, these enzymes differ by their specificity for cleaving the amide bond before or after a specific residue. For complex protein samples, a combination of highly selective proteases has proved to increase proteome coverage by creating complementary peptides (13). 

Olivova, P., Gilar, M., Dorschel, C. A., Gebler, J.C. (2005). Improved peptide identification and protein coverage for proteomic samples using alternative 2D-HPLC MS/MS approaches. ASMS, 2005, San Antonio, TX Poster TP29. 

Figure 20.11 Coverage of protein ErbB2 by shotgun proteomic discovery of sample fixed for various times, including fresh. The color gradient represents the increasing abundance of the peptides. All were identified at an FDR <1%. Reproduced with permission from Reference 20.

Fischer, F., Wolters, D., Rogner, M. and Poetsch, A. (2006) Toward the complete membrane proteome high coverage of integral membrane proteins through transmembrane peptide detection. Mol. Cell. Proteomics 5, 444-453. 

It should be noted that ETD is a relatively inefficient process for doubly protonated peptide precursors [M + 2H]2+, which are the ions most commonly found in bottom-up proteomics experiments. This situation may be retrieved, however, by using a supplemental low-energy CID method (ETciD) to target the nondissociated electron transfer (ET) product, [M + 2H]2+. CID of the ET product then yields c- and z-type fragment ions. Swaney etal.110 have reported that in a large-scale analysis of doubly charged tryptic peptides, the use of ETciD resulted in a median sequence coverage of 89% compared to 63 and 77% for ETD and CID, respectively. 

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