DeMix Workflow for Efficient Identification of Cofragmented Peptides in High Resolution Data-dependent Tandem Mass Spectrometry

Abstract

Based on conventional data-dependent acquisition strategy of shotgun proteomics, we present a new workflow DeMix, which significantly increases the efficiency of peptide identification for in-depth shotgun analysis of complex proteomes. Capitalizing on the high resolution and mass accuracy of Orbitrap-based tandem mass spectrometry, we developed a simple deconvolution method of “cloning” chimeric tandem spectra for cofragmented peptides. Additional to a database search, a simple rescoring scheme utilizes mass accuracy and converts the unwanted cofragmenting events into a surprising advantage of multiplexing. With the combination of cloning and rescoring, we obtained on average nine peptide-spectrum matches per second on a Q-Exactive workbench, whereas the actual MS/MS acquisition rate was close to seven spectra per second. This efficiency boost to 1.24 identified peptides per MS/MS spectrum enabled analysis of over 5000 human proteins in single-dimensional LC-MS/MS shotgun experiments with an only two-hour gradient. These findings suggest a change in the dominant “one MS/MS spectrum - one peptide” paradigm for data acquisition and analysis in shotgun data-dependent proteomics. DeMix also demonstrated higher robustness than conventional approaches in terms of lower variation among the results of consecutive LC-MS/MS runs.

DeMix-Q: Quantification-Centered Data Processing Workflow

“…It is, however, much more pronounced with DDA, regardless of which PIP procedure is used. On the other hand, DDA tends to identify more peptides and give deeper proteome coverage from the same sample than DIA, which is easy to understand, given the burden of peptide identification in DIA from severely convoluted data (2). When the size of comparative proteomics datasets becomes larger, the impact of the missing values becomes progressively worse.…”

“…In this study, we will meet this challenge by introducing a new quantification-centered label-free workflow, DeMix-Q. It represents a LFQ-extension of the previously developed DeMix identification workflow designed for maximizing proteome coverage by identifying co-fragmented peptides (2). But in principle, DeMix-Q does not require DeMix for peptide identification and is compatible with any other peptide identification methods.…”

“…In the popular data-dependent acquisition (DDA) approach named Top-N DDA, the appearance of a peptide-like signal in a "survey" mass spectrum triggers a tandem mass spectrometry (MS/MS) event, targeting the (N) most-abundant precursor ions. Previous studies have shown that, due to the limited speed of a mass spectrometer, the majority of peptide ions detected in MS 1 are not targeted in MS/MS, especially when a nonfractionated complex sample is analyzed (1,2). This low sampling efficiency (Ͻ50%), combined with the stochastic nature of precursor selection and a limited efficiency of MS/MS identification (Ͻ70%) (3), frequently causes the absence of MS/MS identification for an individual peptide in some LC-MS/MS experiments ("runs") within a larger dataset, even when replicate measurements are made (4).…”

Proteogenomics meets cancer immunology: mass spectrometric discovery and analysis of neoantigens

“…Usually, less than a half of these spectra are being attributed to a peptide sequence [14]. Certainly, it is important to increase coverage of understood data in shotgun proteomes.…”

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.…”