Screening for Disulfide Bonds in Proteins by MALDI In-Source Decay and LIFT-TOF/TOF-MS
- 23 August 2002
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 74 (19), 4980-4988
- https://doi.org/10.1021/ac025807j
Abstract
An automated screening method is presented that uses MALDI in-source decay (MALDI-ISD) of disulfide bonds for identification of disulfide-linked peptides in MALDI mass spectra. Peptides released by ISD of a disulfide bond can be detected at an m/z ratio that corresponds to the singly protonated peptide with a reduced cysteine residue. Therefore, screening of peak lists for signal patterns that fulfill the equation, m/z (peak A) + m/z (peak B) − m/z (H2 + H+) = m/z (peak C), facilitated identification of putative ISD fragments of disulfide-linked peptides (peaks A and B) and their precursors (peak C). Signals (peak C) from putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the existence of a disulfide bond. Using this method, we identified all 4 disulfide bonds in RNAseA and 8 two-disulfide clusters comprising 16 out of the 17 disulfide bonds in BSA. The presented screening method accelerated the identification of disulfide bonds in RNAseA and BSA, because the number of MS/MS spectra to be acquired was reduced by 1 order of magnitude. Less than 5% of the signals selected for LIFT-TOF/TOF-MS did not correspond to disulfide-linked peptides. Furthermore, the number of possible assignments for disulfide-linked peptides was reduced by 2−3 orders of magnitude, because knowledge of the mechanism of disulfide bond fragmentation by ISD permitted use of stricter rules for the interpretation of mass spectra. Therefore, interpretation of MS/MS spectra of disulfide-linked peptides was considerably simplified in comparison to conventional approaches.Keywords
This publication has 18 references indexed in Scilit:
- Ideication of disulfide‐linked peptides by isotope profiles produced by peptic digestion of proteins in 50% 18O waterProtein Science, 2001
- Localization of Disulfide Bonds in the Cystine Knot Domain of Human von Willebrand FactorJournal of Biological Chemistry, 2000
- Disulfide Bonds and Protein FoldingBiochemistry, 2000
- Electron Capture Dissociation of Gaseous Multiply-Charged Proteins Is Favored at Disulfide Bonds and Other Sites of High Hydrogen Atom AffinityJournal of the American Chemical Society, 1999
- Determination of Disulfide Structure in Agouti-Related Protein (AGRP) by Stepwise Reduction and AlkylationBiochemistry, 1998
- Metals and Biomolecules -Bioinorganic Analytical ChemistryAnalusis, 1998
- Electron Capture Dissociation of Multiply Charged Protein Cations. A Nonergodic ProcessJournal of the American Chemical Society, 1998
- Cleavage of interchain disulfide bonds following matrix-assisted laser desorptionInternational Journal of Mass Spectrometry and Ion Processes, 1993
- Some Developments in Nuclear Magnetic Resonance of SolidsScience, 1989
- Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltonsAnalytical Chemistry, 1988