on 04-10-201406:50 AM - edited on 10-15-202111:37 AM by AnalyteGuru
Guo X, Trudgian DC, Lemoff A, Yadavalli S, Mirzaei H. Mol Cell Proteomics. 2014 Apr 2. Bottom-up proteomics largely relies on tryptic peptides for protein identification and quantification. Tryptic digestion often provides limited coverage of protein sequence due to issues such as peptide length, ionization efficiency and posttranslational modification (PTM) colocalization. Unfortunately a region of interest in a protein, e.g. due to proximity to an active site or the presence of important PTMs, may not be covered by tryptic peptides. Detection limits, quantification accuracy and isoform differentiation can also be improved with greater sequence coverage. Selected Reaction Monitoring (SRM) would also greatly benefit from being able to identify additional targetable sequences. In an attempt to improve protein sequence coverage and to target regions of proteins that do not generate useful tryptic peptides, we deployed a multi-protease strategy on the HeLa proteome. First, we used seven commercially available enzymes in single, double and triple enzyme combinations. A total of 48 digests were performed. 5223 proteins were detected by analyzing the unfractionated cell lysate digest directly, with 42% mean sequence coverage. Additional strong-anion exchange (SAX) fractionation of the most complementary digests permitted identification of over 3000 more proteins, with improved mean sequence coverage. We then constructed a web application (https://proteomics.swmed.edu/confetti) that allows the community to examine a target protein or protein isoform in order to discover the enzyme or combination of enzymes that would yield peptides spanning a certain region of interest in the sequence. Finally, we examined the utility of non-tryptic digests for SRM. From our SAX data we were able to identify three or more proteotypic SRM candidates within a single digest for 6056 genes. Surprisingly, in 25% of these cases the digest producing the most observable proteotypic peptides was neither trypsin nor Lys-C. SRM analysis of Asp-N vs. tryptic peptides for eight proteins determined that Asp-N yielded higher signal in 5 of 8 cases.