Collision-Induced Dissociation of Lys–Lys Intramolecular Crosslinked Peptides

• Published in: Journal of the American Society for Mass Spectrometry Vol. 20; no. 4; pp. 557 - 566
• Main Authors: Iglesias, Amadeu H., Santos, Luiz F.A., Gozzo, Fabio C.
• Format: Journal Article
• Language: English
• Published: New York Elsevier Inc 01.04.2009; Springer-Verlag; Elsevier; Springer Nature B.V
• Subjects: Aminoacids, peptides. Hormones. Neuropeptides; Analytical Chemistry; Analytical, structural and metabolic biochemistry; Backbone; Bioinformatics; Biological and medical sciences; Biotechnology; Chemistry; Chemistry and Materials Science; Cross-Linking Reagents - chemistry; Crosslinking; Data analysis; Desorption; Fragmentation; Fragments; Fundamental and applied biological sciences. Psychology; Ionization; Ions; Lysine; Lysine - chemistry; Mass spectrometry; Models, Molecular; Organic Chemistry; Peptides; Peptides - analysis; Peptides - chemistry; Proteins; Proteomics; Residues; Search engines; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Succinimides - chemistry; Tandem Mass Spectrometry; Trypsin; Sine Residue; Peptide Crosslinker; Peptide; Modify Peptide; Suberate; Intramolecular reaction; Tandem mass spectrometry; Collisional activation; Analysis method; Peptides; Time of flight method; Cyclic peptides; Polypeptide; Crosslinking; Matrix assisted laser desorption ionization
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1016/j.jasms.2008.11.009

The use of chemical crosslinking is an attractive tool that presents many advantages in the application of mass spectrometry to structural biology. The correct assignment of crosslinked peptides, however, is still a challenge because of the lack of detailed fragmentation studies on resultant species. In this work, the fragmentation patterns of intramolecular crosslinked peptides with disuccinimidyl suberate (DSS) has been devised by using a set of versatile, model peptides that resemble species found in crosslinking experiments with proteins. These peptides contain an acetylated N-terminus followed by a random sequence of residues containing two lysine residues separated by an arginine. After the crosslinking reaction, controlled trypsin digestion yields both intra- and intermolecular crosslinked peptides. In the present study we analyzed the fragmentation of matrix-assisted laser desorption/ionization–generated peptides crosslinked with DSS in which both lysines are found in the same peptide. Fragmentation starts in the linear moiety of the peptide, yielding regular b and y ions. Once it reaches the cyclic portion of the molecule, fragmentation was observed to occur either at the following peptide bond or at the peptide crosslinker amide bond. If the peptide crosslinker bond is cleaved, it fragments as a regular modified peptide, in which the DSS backbone remains attached to the first lysine. This fragmentation pattern resembles the fragmentation of modified peptides and may be identified by common automated search engines using DSS as a modification. If, on the other hand, fragmentation happens at the peptide bond itself, rearrangement of the last crosslinked lysine is observed and a product ion containing the crosslinker backbone and lysine ( m/z 222) is formed. The detailed identification of fragment ions can help the development of softwares devoted to the MS/MS data analysis of crosslinked peptides. The CID of intramolecular crosslinked peptides reveals the fragmentation pathway of these species that are used to study 3D structures of native proteins.