Gas phase ion chemistry of titanium–oxofullerene with ligated solvents

Gas phase fragmentation events of fullerene-like titanium oxo-cluster anions were investigated in detail. The fragmentation channel of the ions was comparable to the fragmentation of C60 ions with systematic C2 losses which is a consequence of topological similarity.

Enhancing Cysteine Chemoproteomic Coverage Through Systematic Assessment of Click Chemistry Product Fragmentation

Mass spectrometry-based chemoproteomics has enabled functional analysis and small molecule screening at thousands of cysteine residues in parallel. Widely adopted chemoproteomic sample preparation workflows rely on the use of pan-cysteine reactive probes such as iodoacetamide alkyne combined with biotinylation via copper-catalyzed azide–alkyne cycloaddition (CuAAC) or ‘click chemistry’ for cysteine capture. Despite considerable advances in both sample preparation and analytical platforms, current techniques only sample a small fraction of all cysteines encoded in the human proteome. Extending the recently introduced labile mode of the MSFragger search engine, here we report an in-depth analysis of cysteine biotinylation via click chemistry (CBCC) reagent gas-phase fragmentation during MS/MS analysis. We find that CBCC conjugates produce both known and novel diagnostic fragments and peptide remainder ions. Among these species, we identified a candidate signature ion for CBCC peptides, the oxonium-biotin fragment ion that is generated upon fragmentation of the N(triazole)–C(alkyl) bond together with cyclization. Guided by our empirical comparison of the fragmentation patterns of five CBCC reagent combinations, we achieved enhanced coverage of cysteine labeled peptides. For larger, fragmentation-prone biotinylation reagents, implementation of labile search afforded unique PSMs and provides a roadmap for the utility of such searches in enhancing chemoproteomic peptide coverage.

Fast Quantitative Analysis of timsTOF PASEF Data with MSFragger and IonQuant

Ion mobility brings an additional dimension of separation to LC–MS, improving identification of peptides and proteins in complex mixtures. A recently introduced timsTOF mass spectrometer (Bruker) couples trapped ion mobility separation to TOF mass analysis. With the parallel accumulation serial fragmentation (PASEF) method, the timsTOF platform achieves promising results, yet analysis of the data generated on this platform represents a major bottleneck. Currently, MaxQuant and PEAKS are most used to analyze these data. However, because of the high complexity of timsTOF PASEF data, both require substantial time to perform even standard tryptic searches. Advanced searches (e.g. with many variable modifications, semi- or non-enzymatic searches, or open searches for post-translational modification discovery) are practically impossible. We have extended our fast peptide identification tool MSFragger to support timsTOF PASEF data, and developed a label-free quantification tool, IonQuant, for fast and accurate 4-D feature extraction and quantification. Using a HeLa data set published by Meier et al. (2018), we demonstrate that MSFragger identifies significantly (∼30%) more unique peptides than MaxQuant (1.6.10.43), and performs comparably or better than PEAKS X+ (∼10% more peptides). IonQuant outperforms both in terms of number of quantified proteins while maintaining good quantification precision and accuracy. Runtime tests show that MSFragger and IonQuant can fully process a typical two-hour PASEF run in under 70 min on a typical desktop (6 CPU cores, 32 GB RAM), significantly faster than other tools. Finally, through semi-enzymatic searching, we significantly increase the number of identified peptides. Within these semi-tryptic identifications, we report evidence of gas-phase fragmentation before MS/MS analysis.