loading . . . In-source fragmentation in mass spectrometry-based proteomics: prevalence, impact, and strategies for mitigation Peptide-level analyses are becoming increasingly popular in mass spectrometry-based proteomics and are being applied, for example, in immunopeptidomics, structural proteomics, and analyses of post-translational modifications. In such analyses, peptides that are not biologically meaningful but instead arise as artifacts prior to mass spectrometry analysis pose the risk of data misinterpretation. Here, we describe an approach based on retention time analysis and precise chromatographic peak matching to identify peptides generated by in-source fragmentation (ISF), which occurs between chromatographic separation of peptide mixtures and the first mass filter of a tandem mass spectrometer (MS). To understand the prevalence and properties of ISF, we generated 13 proteomics datasets and analyzed them along with additional 25 previously published datasets spanning a broad range of sample types, MS, and proteomics approaches including classical bottom-up proteomics, immunopeptidomics, structural proteomics, and phosphoproteomics. We found that, in typical trypsin-digested samples on average 1 % of fully-tryptic peptides and 22 % of semi-tryptic peptides originated from ISF. However, we observed large variations between datasets, and in-source fragments exceeded, in some cases, a third of the total peptide identifications. The extent of ISF was dependent on the peptide sequence, the instrument, method parameters, and sample complexity. Although ISF did not impair relative quantification across samples, it generated peptides that could be misinterpreted qualitatively, inflated peptide identifications, and comprised up to 37 percent of peptides shorter than 9 amino acids in immunopeptidomics datasets. We propose that, for peptide-centric applications, our open-source ISF detection approach be used to re-annotate peptides generated by ISF and remove them to avoid misinterpretation of data. ISF is an increasing concern with improving mass spectrometers, as they enable detection of an ever-increasing number of m/z features, including low abundance features like ISF products. Our work thus addresses a growing issue in proteomics and presents solutions to mitigate the impact of in-source fragment peptides. In the future, improved feature detection algorithms may enable elucidation of new ISF patterns affecting side chains that have been missed so far, which could contribute to explaining the vast space of as-yet unannotated proteomics data. http://dlvr.it/TRnN8h
