Noncovalently associated peptides are observed during liquid chromatography-mass spectrometry and effect cross-link analyses

Cross-linking mass spectrometry draws structural information from covalently linked peptide pairs. When these links do not match to previous structural models, they may indicate changes in protein conformation. Unfortunately, such links can also be the result of experimental error or artefacts. Here, we describe the observation of noncovalently associated peptides during liquid chromatography-mass spectrometry analysis, which can easily be misidentified as crosslinked. Strikingly, they often mismatch to the protein structure. Noncovalently associated peptides presumably form during ionization and can be distinguished from crosslinked peptides by observing coelution of the corresponding linear peptides in MS1, as well as the presence of the individual (intact) peptide fragments in MS2 spectra. To suppress noncovalent peptide formations increasingly disruptive ionization settings can be used, such as in-source fragmentation.