Screening for Disulfide Bonds in Proteins by MALDI In-Source Decay and LIFT-TOF/TOF-MS

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.