Fig. 2: An unbiased workflow to study electrophile selectivity using the MSFragger-based FragPipe computational platform.
From: Profiling the proteome-wide selectivity of diverse electrophiles
a, Labelling of the proteome of S. aureus SH1000 with 1 mM IA‑alkyne and analysis using the isoDTB-ABPP workflow5 (Fig. 1b) resulted in generation of MS data for analysis of proteome-wide reactivity and selectivity. b, Through analysis with an open search in MSFragger31,32, the masses of modification that occurred proteome-wide were assigned. The peaks highlighted in red are the expected modifications (Δmexp) resulting from alkylation and modification with the light and heavy isoDTB tags, respectively. Further modifications of the alkylated peptides by oxidation (Δmox), formylation (Δmf) or carbamidomethylation on a second cysteine (ΔmCAM) were also detected. c, One peak pair (Δmexp) was selected for an MSFragger mass offset search to localize this modification to the modified amino acid(s), allowing selectivity to be assessed across all proteinogenic amino acids. The bar graph represents the fraction of all modified sites that were modified at the indicated amino acid. The same data are also presented in a letter plot, in which the size of the letter is scaled by the fraction of all modified sites that were modified at the indicated amino acid. All amino acids that were modified in fewer than 5% of cases are summarized as ‘Others’. The total number of modified sites is given as a bar graph on top of the letter plot. d, One amino acid (cysteine) was selected for quantification at the selected masses (Δmexp) using MSFragger closed search and the IonQuant quantification module42. Here, two datasets were analysed, in which the heavy and light samples were mixed at a ratio of 1:1 and 4:1, respectively. Grey solid lines indicate the expected values of log2(R) = 0 and log2(R) = 2. Grey dashed lines indicate the respective preferred window of quantification (−1 < log2(R) < 1 for the 1:1 ratio; 1 < log2(R) < 3 for the 4:1 ratio). The total number of quantified sites is given at the top of the plot. The mass spectrometric experiments used for this analysis were performed as part of an earlier study5. All data are based on technical duplicates. C-term, C-terminal modification; N-term, N-terminal modification; PSM, peptide spectrum match.
