Improving protein identification sensitivity by combining MS and MS/MS information for shotgun proteomics using LTQ-Orbitrap high mass accuracy data

Abstract

We investigated and compared three approaches for shotgun protein identification by combining MS and MS/MS information using LTQ-Orbitrap high mass accuracy data. In the first approach, we employed a unique mass identifier method where MS peaks matched to peptides predicted from proteins identified from an MS/MS database search are first subtracted before using the MS peaks as unique mass identifiers for protein identification. In the second method, we used an accurate mass and time tag method by building a potential mass and retention time database from previous MudPIT analyses. For the third method, we used a peptide mass fingerprinting-like approach in combination with a randomized database for protein identification. We show that we can improve protein identification sensitivity for low-abundance proteins by combining MS and MS/MS information. Furthermore, "one-hit wonders" from MS/MS database searching can be further substantiated by MS information and the approach improves the identification of low-abundance proteins. The advantages and disadvantages for the three approaches are then discussed.

Figure 1

Scoring MZ Hits using randomized databases (Replicate 1). Blue solid line, distribution of z scores computed from unique MZ hits to the randomized yeast database R₂; Red dash line, distribution of z scores computed from unique MZ hits to the yeast database (target database).

Figure 2

Effect of retention time tolerance on protein identification using the accurate mass and time tag approach.