Application of MALDI TOF/TOF mass spectrometry and collision‐induced dissociation for the identification of disulfide‐bonded peptides

Abstract

This paper describes a method for the fast identification and composition of disulfide-bonded peptides. A unique fragmentation signature of inter-disulfide-bonded peptides is detected using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry and high-energy collision-induced dissociation (CID). This fragmentation pattern identifies peptides with an interconnected disulfide bond and provides information regarding the composition of the peptides involved in the pairing. The distinctive signature produced using CID is a triplet of ions resulting from the cleavage of the disulfide bond to produce dehydroalanine, cysteine or thiocysteine product ions. This method is not applicable to intra-peptide disulfide bonds, as the cleavage mechanism is not the same and a triplet pattern is not observed. This method has been successfully applied to identifying disulfide-bonded peptides in a number of control digestions, as well as study samples where disulfide bond networks were postulated and/or unknown.

Mass spectrometry-based strategies for protein disulfide bond identification

“…In addition to the typical b/y ions from individual peptide chains, the b/y ions containing cysteine residue, cysteine thioaldehyde (-2 Da), cysteine persulfide (+32 Da), or dehydroalanine (-34 Da) due to the breakage of S-S or C-S bond can also be observed Kaltashov 2006, Choi et al 2010), as shown in Figure 2, and so are those fragment ions linked by intact disulfide bonds. Similar phenomenon was reported in the high-energy CID mode in MALDI TOF/TOF instrument (Janecki and Nemeth 2011). It is labor intensive and time consuming to examine all the MS/MS spectra for all possible cysteinyl peptide combinations due to spectra complexity.…”

Tandem Mass Spectrometry of Peptides

“…They identified fragments for low-charged protein precursor ions that correspond to the breakage of disulfide-bonds and of protein backbone. These related disulfide-bond fragments resulted from the addition or subtraction of a hydrogen atom or sulfhydryl group with mass changes of -32, -2, +2 and +32 Da for -SH, -H, +H, and +SH, respectively, similar to the patterns obtained by Janecki & Nemeth (2011) for tryptic peptides.…”

“…The conventional methodology for studying this PTM is, with HPLC or mass spectrometry, to compare protein enzymatic digests using the target protein in their reduced and native forms, where the chromatographic peaks or masses obtained are compared and the differences obtained in the native form are considered due to possible disulfide bonded peptides. In a recent study by Janecki & Nemeth (2011) an efficient method using MALDI-TOF-TOF and high-energy CID was applied to identify disulfide-bonded peptides in proteins with well-documented disulfide bond networks (namely bovine insulin and human serum albumin) and on recombinant proteins where disulfide bonds were not defined, without previous separation of the protein native digests. This method was based on the fact that a number of fragmentation processes happen around the S-S bond leading to a "triplet peak" signature in the spectrum (Fig.…”

“…4), which results from the symmetric cleavage of the S-S bond originating a cysteine fragment (middle peak of the "triplet peak signature") and asymmetric cleavage of the S-S bond originating dehydroalanine and thiocysteine fragments with a mass difference from the middle peak of -34 and +32 Da, respectively ( Fig. 4; Janecki & Nemeth, 2011). Sometimes a smaller peak identified as the dehydrocysteine peak can occur around the cysteine m/z peak.…”

Identification of sulfenylated cysteines inArabidopsis thalianaproteins using a disulfide-linked peptide reporter

“…+S, m/z 940.37) and a cysteine-to-dehydroalanine conversion (green; -SH2, m/z 874.41) are consistently present in C598SEIWDR CL PSMs (Figure 3D and E). This distinctive pattern of triplet ions is characteristic for inter-protein disulfides (Janecki and Nemeth, 2011) and used, for instance, by dedicated disulfide CL identification algorithms, such as DBond and MS2DB+ (Murad et al, 2011). Together with the C598SEIWDR y fragment ions, these precursor triplet ions form a distinctive C598SEIWDR fragment ion fingerprint.…”