Alpha-XIC: a deep neural network for scoring the coelution of peak groups improves peptide identification by data-independent acquisition mass spectrometry

ABSTRACTMotivationThe peptide-centric identification methodologies of data-independent acquisition (DIA) data mainly rely on scores for the mass spectrometric signals of targeted peptides. Among these scores, the coelution scores of peak groups constructed by the chromatograms of peptide fragment ions have a significant influence on the identification. Most of the existing coelution scores are achieved by artificially designing some functions in terms of the shape similarity, retention time shift of peak groups. However, these scores cannot characterize the coelution robustly when the peak group is in the circumstance of interference.ResultsOn the basis that the neural network is more powerful to learn the implicit features of data robustly from a large number of samples, and thus minimizing the influence of data noise, in this work, we propose Alpha-XIC, a neural network-based model to score the coelution. By learning the characteristics of the coelution of peak groups derived from identified peptides, Alpha-XIC is capable of reporting robust coelution scores even for peak groups with interference. With this score appending to initial scores generated by the accompanying identification engine, the ensuing statistical validation tool can update the identification result and recover the misidentified peptides. In our evaluation of the HeLa dataset with gradient lengths ranging from 0.5h to 2h, Alpha-XIC delivered 16.7% ~ 49.1% improvements in the number of identified precursors at 1% FDR. Furthermore, Alpha-XIC was tested on LFQbench, a mixed-species dataset with known ratios, and increased the number of peptides and proteins fell within valid ratios by up to 16.6% and 13.8%, respectively, compared to the initial identification.Availability and ImplementationSource code are available at www.github.com/YuAirLab/Alpha-XIC.

Evaluation of a Dual Isolation Width Acquisition (DIWA) method for isobaric labelling ratio decompression

ABSRACTIsobaric labelling is a highly precise approach for protein quantification. However, due to the isolation interference problem, isobaric tagging suffers from ratio underestimation at the MS2 level. The use of narrow isolation widths is a rational approach to alleviate the interference problem; however, this approach compromises proteome coverage. We reasoned that although a very narrow isolation window will result in loss of peptide fragment ions, the reporter ion signals will be retained for a significant portion of the spectra. Based on this assumption we have designed a Dual Isolation Width Acquisition (DIWA) method, in which each precursor is first fragmented with HCD using a standard isolation width for peptide identification and preliminary quantification, followed by a second MS2 HCD scan using a much narrower isolation width for the acquisition of quantitative spectra with reduced interference. We leverage the quantification obtained by the “narrow” scans to build linear regression models and apply these to decompress the fold-changes measured at the “standard” scans. We evaluate the DIWA approach using a nested two species/gene knockout TMT-6plex experimental design and discuss the perspectives of this approach.