Impact of Multiple S‐Palmitoylation on Peptide Ionization and Fragmentation in Mass Spectrometry

Lipidation, such as S‐palmitoylation, is an important, reversible post‐translational modification of proteins determining not only their stability and folding but also their interactions with other proteins or membranes. However, in contrast to other post‐translational modifications, lipidation is less explored, and lipidated proteins are underrepresented in large‐scale studies. To advance the analysis of S‐palmitoylation by mass spectrometry (MS), a model peptide containing four potential modification sites is selected. By selectively introducing S‐palmitoylation, a set of multiply modified peptides is generated, differing in the sites as well as the degree of modification. Importantly, the solubility of the peptides decreased tremendously with increasing degree of modification, requiring the use of alternative solvents. Nonetheless, using direct‐infusion MS, the ionization and fragmentation behavior of the differently modified peptides is characterized. Lipidation is found to be stable during tandem MS, and the sites of modification can be unambiguously identified. The use of dimethyl sulfoxide during electrospray ionization further improves the signal intensity of multiply modified peptides. In summary, the identification of S‐palmitoylation even in multiply modified peptides is possible; however, further improvements are required for large‐scale analyses.