Monday, May 13, 2013

Multiple enzymes support in PEAKS - Full Protein Coverage

PEAKS 6 introduced a new feature specifically targeting the experiments that use multiple enzyme digestions to increase protein coverage.

In the past, users have to search each sample separately and combine all the results manually afterwards or using none enzyme option to analyze all samples in one go which may cause higher false positives. Now in PEAKS, users can specify enzyme for each sample when creating a multi-sample project. Then in de novo sequencing and PEAKS DB, user can choose 'sample enzyme' in the enzyme list as the search option. PEAKS will use the correct enzyme when analyzing each sample.

From our users feedback, this feature is extremely useful when you want to fully characterize a single protein.The following example shows how big a difference this feature may make.

ALBU_BOVIN protein ordered from a reputable vendor was digested with Trypsin, LysC, GluC. The dataset is generated from Thermo Orbitrap instrument. Three searches were performed. The first one uses inChorus function to launch Mascot search (version 1.4) on the trypsin sample only. The second search uses standard PEAKS DB search on the trypsin sample. The third search uses the complete analysis workflow, including PEAKS PTM and SPIDER, on all three samples and uses "sample enzyme" as the enzyme option. The results are all filtered to only keep the very confident PSMs at 0.1% FDR level.

Mascot and PEAKS DB are able to achieve 73% and 86% protein coverage using only the trypsin sample respectively. In the protein coverage view below, the blue bars are the PSMs that matched the protein sequence at that position.

Mascot result on the trypsin sample
PEAKS DB result on the trypsin sample

PEAKS complete analysis on all three samples reported 96% coverage on the protein. The uncovered 4% is in the protein N-terminal region, which is most likely cleaved-off and not in the purchased sample1.
PEAKS complete analysis result on all three samples

1specific binding site (Asp-Thr-His-Lys) for Cu(II) ions. T. Peters Jr., F.A. Blumenstock. J. Biol. Chem., 242 (1967), p. 1574

1 comment:

  1. Charles River has extensive experience in developing and establishing protein characterization methods, particularly pharmacopeial and product-specific in Protein Characterization Services.