title: A pseudo MS3 approach for identification of disulfide-bonded proteins: uncommon product ions and database search.
abstract: 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 MS(3) 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 c(i-1) ions (the i(th) 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 MS(3) 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 MS(3) 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.
authors.affiliation: Applied Biotechnology Branch, Air Force Research Laboratory, Dayton, OH 45433, USA. jianzhongc@yahoo.com
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Chen
authors.forename: Pavel
authors.initials: P
authors.lastname: Shiyanov
authors.forename: John J
authors.initials: JJ
authors.lastname: Schlager
authors.forename: Kari B
authors.initials: KB
authors.lastname: Green
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D030562:Databases, Protein; D004220:Disulfides / Q000737:chemistry*; D007477:Ions / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004220:Disulfides; D007477:Ions; D011506:Proteins; D009113:Muramidase