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.
Highlights
From the ‡Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden; §Biomotif AB, Stockholm SE-182 12, Sweden
According to the prevailing assumption of “one MS/MS spectrum– one peptide,” chimeric MS/MS spectra are generally unwelcome in dependent acquisition (DDA), because the product ions from different precursors may interfere with the assignment of MS/MS fragment identities, increasing the rate of false discoveries in database search [8, 9]
We used The OpenMS Proteomics Pipeline (TOPP) to create this feature map, assembling full MS spectra into peptide features based on isotopic modeling and chromatographic tracing [20, 24]
Summary
From the ‡Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden; §Biomotif AB, Stockholm SE-182 12, Sweden. In a typical LC-MS analysis of unfractionated human cell lysate, over 100,000 individual peptide isotopic patterns can be detected [4], which corresponds to simultaneous elution of hundreds of peptides With this complexity, a mass spectrometer needs to achieve Ն25 Hz MS/MS acquisition rate to fully sample all the detectable peptides, and Ն17 Hz to cover reasonably abundant ones [4]. The “second peptide identification” method implemented in Andromeda/MaxQuant workflow [16] submits the same dataset to the search engine several times based on the list of chromatographic peptide features, subtracting assigned MS/MS peaks after each identification round. This approach is similar to the ProbIDTree search engine that performed iterative identification while removing assigned peaks after each round of identification [17]
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