Gas-phase scrambling of disulfide bonds during matrix-assisted laser desorption/ionization mass spectrometry analysis

Abstract

Evidence for photo-induced radical disulfide bond scrambling in the gas phase during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described. The phenomenon was observed during the analysis of tryptic peptides from insulin and was confirmed in the determination of disulfide bonds in the rhamnose-binding lectin SEL24K from the Chinook salmon Oncorhynchus tshawytscha. A possible mechanism for this surprising scrambling is proposed. Despite this finding, the disulfide bond pattern in SEL24K was assigned unambiguously by a multi-enzyme digestion strategy in combination with MALDI mass spectrometry. The pattern was found to be symmetrical in the tandem repeat sequence of SEL24K. To the best of our knowledge, this is the first report of disulfide bond scrambling in the gas phase during MALDI-MS analysis. This observation has important ramifications for unambiguous assignment of disulfide bonds. (J Am Soc

Disulfide bond decay during matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry experiments

“…We measured peptide C2 in oxidized form (with intramolecular disulfide bonds) as well as non-reduced insulin (with intermolecular disulfide bonds) in various DHB matrix isomers (see below) with different laser intensity, and we never observed peaks shifted by 2 Da compared to the original oxidized molecules. In our opinion, which is consistent with other references, [10] the answer to the question in the title of this paragraph should be 'No!'. Mass spectra of cysteine-containing peptides modified by Ellman's reagent Mass spectra of the peptide C2 modified by Ellman's reagent (structure C2-TNB 2 ) showed that this modification is not stable under MALDI conditions.…”

“…We measured peptide C2 in oxidized form (with intramolecular disulfide bonds) as well as non‐reduced insulin (with intermolecular disulfide bonds) in various DHB matrix isomers (see below) with different laser intensity, and we never observed peaks shifted by 2 Da compared to the original oxidized molecules. In our opinion, which is consistent with other references, the answer to the question in the title of this paragraph should be ‘No!’.…”

“…[8] The disulfide bond can also be the target of reduction by DHB ( Fig. 1(a)); Works by Zhang and Knochenmuss [9] or Zhao et al [10] suggest that the disulfide reduction during the desorption/ionization process proceeds in the gas phase. However, there are other papers that deny the reductive effect of DHB matrix.…”

Bond-Specific Dissociation Following Excitation Energy Transfer for Distance Constraint Determination in the Gas Phase

“…Although the charge state per se may not be important, it is likely for peptide pairs that charge repulsion overcomes any noncovalent interactions that otherwise inhibit dissociation. In the absence of Coulombic repulsion, two peptide radicals generated by disulfide bond cleavage can simply recombine 38 to regenerate the disulfide bond if the fragments are temporarily held together by noncovalent interactions.…”

Diversified Carbohydrate-Binding Lectins from Marine Resources

“… Figure 3 shows the aligned amino acid sequences of various RBL-CRDs. The disulfide bond pairings of RBLs have been determined for Spanish mackerel lectin (SML) by protein sequencing combined with peptide mapping [59] and for SEL24K from Chinook salmon by matrix-assisted laser desorption/ionization (MALDI) mass-spectrometry [132], respectively. Each RBL-CRD had the same disulfide bonding patterns: Cys(1)–Cys(3), Cys(2)–Cys(8), Cys(4)–Cys(7), and Cys(5)-Cys(6) (Figure 3).…”