A Pseudo MS^sup 3^ Approach for Identification of Disulfide-Bonded Proteins: Uncommon Product Ions and Database Search

• Published in: Journal of the American Society for Mass Spectrometry Vol. 23; no. 2; p. 225
• Main Authors: Chen, Jianzhong, Shiyanov, Pavel, Schlager, John J, Green, Kari B
• Format: Journal Article
• Language: English
• Published: New York Springer Nature B.V 01.02.2012
• Subjects: Aspartic acid; Chemical bonds; Ionization; Ions; Lysine; Lysozyme; Mass spectrometry; Peptides; Proteins; Quadrupoles; Searching; Spectra
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1007/s13361-011-0294-6

It has previously been reported that disulfide and backbone bonds of native intact proteins can be concurrently cleaved using electrospray ionization (ESI) and collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). However, the cleavages of disulfide bonds result in different cysteine modifications in product ions, making it difficult to identify the disulfide-bonded proteins via database search. To solve this identification problem, we have developed a pseudo MS3 approach by combining nozzle-skimmer dissociation (NSD) and CID on a quadrupole time-of-flight (Q-TOF) mass spectrometer using chicken lysozyme as a model. Although many of the product ions were similar to those typically seen in MS/MS spectra of enzymatically derived peptides, additional uncommon product ions were detected including ci-1 ions (the ith residue being aspartic acid, arginine, lysine and dehydroalanine) as well as those from a scrambled sequence. The formation of these uncommon types of product ions, likely caused by the lack of mobile protons, were proposed to involve bond rearrangements via a six-membered ring transition state and/or salt bridge(s). A search of 20 pseudo MS3 spectra against the Gallus gallus (chicken) database using Batch-Tag, a program originally designed for bottom up MS/MS analysis, identified chicken lysozyme as the only hit with the expectation values less than 0.02 for 12 of the spectra. The pseudo MS3 approach may help to identify disulfide-bonded proteins and determine the associated post-translational modifications (PTMs); the confidence in the identification may be improved by incorporating the fragmentation characteristics into currently available search programs.