Noncovalently Associated Peptides Observed during Liquid Chromatography-Mass Spectrometry and Their Effect on Cross-Link Analyses

• Published in: Analytical chemistry (Washington) Vol. 91; no. 4; pp. 2678 - 2685
• Main Authors: Giese, Sven H, Belsom, Adam, Sinn, Ludwig, Fischer, Lutz, Rappsilber, Juri
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 19.02.2019
• Subjects: Amino Acid Sequence; Analytical chemistry; Animals; Chemistry; Chickens; Chromatography; Chromatography, Liquid; Conalbumin - analysis; Conalbumin - chemistry; Conalbumin - metabolism; Creatine Kinase - analysis; Creatine Kinase - chemistry; Creatine Kinase - metabolism; Cross-Linking Reagents - chemistry; Crosslinking; Horses; Humans; Ionization; Ions; Liquid chromatography; Mass Spectrometry; Mass spectroscopy; Myoglobin - analysis; Myoglobin - chemistry; Myoglobin - metabolism; Peptides; Peptides - analysis; Peptides - chemistry; Peptides - metabolism; Protein Multimerization; Protein structure; Proteins; Rabbits; Scientific imaging; Serum Albumin, Human - analysis; Serum Albumin, Human - chemistry; Serum Albumin, Human - metabolism; Spectroscopy; Structural models
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/acs.analchem.8b04037

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 artifacts. Here, we describe the observation of noncovalently associated peptides during liquid chromatography-mass spectrometry analysis, which can easily be misidentified as cross-linked. Strikingly, they often mismatch to the protein structure. Noncovalently associated peptides presumably form during ionization and can be distinguished from cross-linked peptides by observing coelution of the corresponding linear peptides in MS1 spectra, 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.