Non-covalently-associated peptides are observed during liquid chromatography-mass spectrometry and affect crosslink analyses

Abstract

Crosslinking 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 non-covalently-associated peptides during liquid chromatography-mass spectrometry analysis, which can easily be misidentified as crosslinked. Strikingly, they often mismatch to the protein structure. Non-covalently-associated peptides presumably form during ionization and can be distinguished from crosslinked peptides by observing co-elution of the corresponding linear peptides in MS1, as well as the presence of the individual (intact) peptide fragments in MS2 spectra. To suppress non-covalent peptide formations increasingly disruptive ionization settings can be used, such as in-source fragmentation.