Mapping Cell Surface Proteolysis with Plasma Membrane-Targeted Subtiligase

N terminomics methods combine selective isolation of protein N-terminal peptides with mass spectrometry (MS)-based proteomics for global profiling of proteolytic cleavage sites. However, traditional N terminomics workflows require cell lysis before N-terminal enrichment and provide poor coverage of N termini derived from cell surface proteins. Here, we describe application of subtiligase-TM, a plasma membrane-targeted peptide ligase, for selective biotinylation of cell surface N termini, enabling their enrichment and analysis by liquid chromatography-tandem MS (LC-MS/MS). This method provides increased coverage of and specificity for cell surface N termini and is compatible with existing quantitative LC-MS/MS workflows.