Precursor-Ion Mass Re-Estimation Improves Peptide Identification on Hybrid Instruments

Abstract

Mass spectrometry-based proteomics experiments have become an important tool for studying biological systems. Identifying the proteins in complex mixtures by assigning peptide fragmentation spectra to peptide sequences is an important step in the proteomics process. The 1–2 ppm mass-accuracy of hybrid instruments, like the LTQ-FT, has been cited as a key factor in their ability to identify a larger number of peptides with greater confidence than competing instruments. However, in replicate experiments of an 18-protein mixture, we note parent masses deviate 171 ppm, on average, for ion-trap data directed identifications and 8 ppm, on average, for preview Fourier transform (FT) data directed identifications. These deviations are neither caused by poor calibration nor by excessive ion-loading and are most likely due to errors in parent mass estimation. To improve these deviations, we introduce msPrefix, a program to re-estimate a peptide’s parent mass from an associated high-accuracy full-scan survey spectrum. In 18-protein mixture experiments, msPrefix parent mass estimates deviate only 1 ppm, on average, from the identified peptides. In a cell lysate experiment searched with a tolerance of 50 ppm, 2295 peptides were confidently identified using native data and 4560 using msPrefixed data. Likewise, in a plasma experiment searched with a tolerance of 50 ppm, 326 peptides were identified using native data and 1216 using msPrefixed data. msPrefix is also able to determine which MS/MS spectra were possibly derived from multiple precursor ions. In complex mixture experiments, we demonstrate that more than 50% of triggered MS/MS may have had multiple precursor ions and note that spectra with multiple candidate ions are less likely to result in an identification using TANDEM. These results demonstrate integration of msPrefix into traditional shotgun proteomics workflows significantly improves identification results.

MixGF: Spectral Probabilities for Mixture Spectra from more than One Peptide

“…This shows that as the complexity of the sample increases, more peptides with similar precursors will co-elute at same retention time and thus mixture spectra indeed constitute a significant fraction of all MS/MS spectra in the data. Such observations have been reported in studies showing that multiple precursors often coincide within the same MS/MS precursor isolation window for as many as 50% of all MS/MS spectra (5,4). But while it remains unclear what fraction of these cofragmented peptides is identifiable, here we show that a large number of co-eluting peptides can be identified.…”

“…However, it is increasingly being recognized that this assumption that each MS/MS spectrum comes from only one peptide is often not valid. Several recent analyses show that as many as 50% of the MS/MS spectra collected in typical proteomics experiments come from more than one peptide precursor (4,5). The presence of multiple peptides in mixture spectra can decrease their identification rate to as low as one half of that for MS/MS spectra generated from only one peptide (6,7,8).…”

“…In particular, we show how to extend the generating approach for mixture from two peptides. We focus on this relatively simple case of mixture spectra because it accounts for a large fraction of mixture spectra presented in traditional DDA workflows (5). This allows us to test and develop algorithmic concepts using readily-available DDA data because data with more complex mixture spectra such as those from DIA workflows (11) is still not widely available in public repositories.…”

Comprehensive Absolute Quantification of the Cytosolic Proteome of Bacillus subtilis by Data Independent, Parallel Fragmentation in Liquid Chromatography/Mass Spectrometry (LC/MSE)

“…Commonly applied data dependent acquisition (DDA) methods for peptide identification suffer from some limitations. Often low abundant peptides with a low MS signal intensity are discriminated and their isobaric precursor ions cannot be isolated leading to low scores in database search and wrong assignments (12,13). These obstacles lead to lower protein sequence coverage in general and higher numbers of protein identifications based on a single peptide only.…”

Peptide-Centric Proteome Analysis: An Alternative Strategy for the Analysis of Tandem Mass Spectrometry Data

“…Considerations for Mixture Spectra-When investigating a complex proteome with shotgun proteomics, mixture spectra are a common occurrence. Although conventional DDA uses narrow isolation windows (typically ϳ2 m/z-wide) targeting single precursor ion species for fragmentation, as many as 50% of the MS/MS spectra are mixed (35,39,49). The frequency and impact of mixture spectra in a DDA experiment vary with the sample complexity, LC separation, acquisition parameters, and instrumentation.…”