MassSpectrum Analyzer: An interactive platform for proteomic searching parameter refinement and peptide modification focused re-scoring

Abstract

Peptide spectrum annotation is critical for the assignment of peptides and the localisation of modifications. While many existing tools provide spectrum annotation capacities, they often lack the flexibility required to allow bespoke spectral annotation of peptides containing multiple labile modifications or the accurate assignment of peptides in which fragmentation deviates from canonical patterns. In these cases, user-guided annotation is widely used to improve assignment completeness, however it typically does not integrate peptide scoring, making it challenging to assess the empirical improvement of the associated annotation and its impact on downstream false-discovery rate estimations. Here, we introduce an interactive annotation environment, the "MassSpectrum Analyzer", which aims to streamline the exploration and analysis of modified peptides by enabling user-defined customisation with peptide scoring. Using (2-Aminoethyl)trimethylammonium carboxyl-derivatised peptides and glycopeptides as case studies we demonstrate the capacity of the MassSpectrum Analyzer to rapidly explore and allow the assessment of modified peptide datasets. By enabling direct assessment of the impact of user-guided choices on peptide scoring, we show how the detection of highly modified peptides can be improved through post-search integration of modification fragmentation information in a statistically robust manner. Similarly, by permitting comparisons of peptide ion intensities across spectra, we show that global fragmentation patterns can be quantified allowing the interrogation of trends that only become clear when spectra are assessed en masse. Combined, the MassSpectrum Analyzer streamlines the generation of publication-ready spectra and provides a means to assess how the inclusion of annotated features influences assignment scores.