Identification-Free Control over the Precursor Isotopic Mass Misassignment in Orbitrap-Based Proteomics.

Selection of a precursor ion from a peptide isotopic cluster to obtain a fragmentation mass spectrum is a crucial step in data-dependent proteome analysis. However, the monoisotopic mass assignment performed in this step is often an issue confronted by the data acquisition software of hybrid Orbitrap FTMS that is most widely used in proteomics. To address the problem, many data processing tools, such as raw data converters and search engines, have optional accounting for the precursor mass shift due to the isotopic error. These solutions require additional data preprocessing steps and lead to an increase in the search space, thus making the analysis longer and/or less reliable. In this work, we processed 100 Orbitrap-based LC-MS/MS runs from 10 publicly available data sets to examine the rate of precursor isotope misassignment. The effect from taking the isotope error into account during the search on the number of identified peptides varied in a wide range from 0 to 33%. Thus, it may be tempting to spend extra time before or during a search to account for the mass assignment issue. Alternatively, this effect can be predicted using an identification-free metric, which can be a part of data quality control software. Based on the results obtained in this work, we propose such a metric be further added into the visual and intuitive quality control software, viQC, developed previously and available at https://github.com/lisavetasol/viQC. It takes about a minute to calculate and plot nine quality metrics, including the proposed one for typical proteome analysis.