Recent advances have rekindled the interest in ion mobility spectrometry as an additional dimension of separation in mass spectrometry (MS)– based proteomics. It separates ions according to their size and shape in the gas phase. Here, we set out to investigate the effect of 22 different post-translational modifications (PTMs) on the collision cross section (CCS) of peptides. In total, we analyzed about 4700 pairs of matching modified and unmodified peptide ions by trapped ion mobility spectrometry (TIMS). Linear alignment based on spike-in reference peptides resulted in highly reproducible CCS values with a median coefficient of variation of 0.3%. On a global level, we observed a redistribution in the m/z vs. ion mobility space for modified peptides upon changes in their charge state. Pairwise comparison between modified and unmodified peptides of the same charge state revealed median shifts in CCS between −1.1% (lysine formylation) and +4.5% (O-GlcNAcylation). In general, increasing modified peptide masses were correlated with higher CCS values, in particular within homologous PTM series. However, investigating the ion populations in more detail, we found that the change in CCS can vary substantially for a given PTM depending on the gas phase structure of its unmodified counterpart. In conclusion, our study shows PTM– and sequence–specific effects on the cross section of peptides, which could be further leveraged for proteome–wide PTM analysis.

Mass spectrometry has revolutionized cell signaling research by vastly simplifying the identification and quantification of many thousands of phosphorylation sites in the human proteome. Defining the cellular response to internal or external perturbations in space and time is crucial for further illuminating functionality of the phosphoproteome. Here we describe µPhos, an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96–well cell culture experiments in < 8 hours total processing time. By minimizing transfer steps and reducing liquid volumes to < 200 µL, we demonstrate increased sensitivity, over 90% selectivity, and excellent quantitative reproducibility. Employing highly sensitive trapped ion mobility mass spectrometry, we quantify more than 20,000 unique phosphopeptides in a human cancer cell line using 20 µg starting material, and confidently localize > 5,000 phosphorylation sites from 5 µg. This depth covers key intracellular signaling pathways, rendering sample-limited applications and extensive perturbation experiments with hundreds of samples viable.