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

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.

MALDI In-Source Decay, from Sequencing to Imaging

“…Indeed, a disulfide bond tends to be reduced during the ISD process [46] and therefore acts as a quencher of the fragmentation of neighboring amino acids. This observation was exploited by Schnaible et al to localize disulfide bonds on HPLC-separated peptides [47,48]. Moreover, PTMs that do not interfere with the ISD fragmentation of the peptide backbone can be studied by ISD as well.…”

A new method for analysis of disulfide-containing proteins by matrix-assisted laser desorption ionization (MALDI) mass spectrometry

“…However, in the interpretation of the obtained data, one must exercise extreme caution because 1,5-DAN can dramatically enhance in-source decay (ISD) fragmentation [16]. Recently, a screening method that uses ISD of disulfide bonds for identification of disulfidelinked peptides in mass spectra has been developed [17,18]. Zhang and Kaltashov reported a method to detect disulfide-linked proteins using the broadband collisionactivated dissociation (CAD) in the negative ion mode [19].…”

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination

“…Partial reductive cyanylation was previously described to circumvent this limitation, 37 however, we performed manual identification of the multiply cross-linked peptides in order to avoid unwanted chemical modifications. The manual MS/MS data analysis was greatly simplified by the predominance of singly-charged precursor ions in the MALDI spectra, and we identified a list of IgG2 disulfide isoform-specific peptides that will be useful for future IgG2 disulfide isoform assessments.…”

“…26,37 Briefly, partial reduction in the ion source results in the detection of two reduced peptides (P 1 -SH and P 2 -SH) in addition to the original DSB peptide (P 1 -S-S-P 2 ). The m/z of the DSB peptide is therefore the sum of the m/z values of P 1 -SH and P 2 -SH, minus the molecular weight of (H 2  + H) + (Figure 2).…”