Sulfopeptide fragmentation in electron-capture and electron-transfer dissociation

Abstract

Sulfopeptides can be misassigned as phosphopeptides because of the isobaric nature of the sulfo-and the phosphomoieties. Instruments having the ability to measure mass with high accuracy may be employed to distinguish these moieties based on their mass defect (the sulfo-group is 9 mmu lighter than the phosphomoiety). However, the assignment of the exact site(s) of post-translational modification is required to probe biological function. We have reported earlier that peptides with identical sequences containing either O-sulfo-or Ophospho-modifications display different fragmentation behavior (K. F. Medzihradszky et al., Mol. Cell. Proteom. 2004, 3, 429 -440). We have also established that O-sulfo moieties are susceptible to side-chain fragmentation during collision-induced dissociation. Our present study provides evidence that neutral SO 3 losses can also occur in electron capture dissociation and electron-transfer dissociation experiments. We also report that such neutral losses may be reduced by fragmenting peptide-alkali metal adducts, such as sodiated or potassiated peptides. ( -5]. Sulfopeptides also display an 80 Da mass increase as well as similar chromatographic and mass spectrometric behavior [6]. The sulfopeptides are 9 mmu lighter than their phosphorylated counterparts. They can easily be misidentified as phosphopeptides. However, the most significant difference between phosphoand sulfopeptides is revealed during MS/MS analysis. In CID analyses phosphorylated Tyr residues retain the modification, and even their modified immonium ion is sometimes observed, while phosphorylated Ser and Thr residues undergo ␤-elimination of H 3 PO 4 . Thus, the fragments detected in the CID spectra of phosphopeptides either display a ϩ80 Da or a Ϫ18 Da mass shift in comparison to the fragments of an unmodified peptide. Under identical conditions, collisional activation of Osulfopeptides leads to a gas-phase rearrangement reaction that completely eliminates the sulfate from the molecular ion, and the CID fragment spectrum is practically identical to that observed for the unmodified molecule [6]. We have analyzed numerous O-sulfopeptides and have not detected any CID fragments that still contained the sulfate group.While the different CID behavior proved to be a reliable tool for the differentiation of these two isobaric modifications, the gas-phase elimination of the sulfomoiety also prevented the assignment of the modification site. Therefore, it seemed essential to evaluate electron-capture dissociation based on reports that most of the labile side chains of peptides and proteins studied remain intact [7]. Electron-transfer dissociation [8,9] was also employed to analyze synthetic phospho-and sulfopeptides. Here we report our observations on the ECD and ETD fragmentation of sulfopeptides. Experimental MaterialsPhosphopeptides and sulfopeptides were synthesized as described earlier [6]. Their sequences are as follows: RIEVALsTK; RIEVALpTK; LAGLQDEIGsSLR; LAGLQDEIGpSLR; Ac-LAGLQDEIGsSLR-amide. Caerulein, ϽQQDsYTGWMDF-ami...

Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii

“…There has been some discussion about the misidentification of phospho-and sulfopeptides because sulfopeptides also display an 80-Da mass increase (22). There are several reasons that render it unlikely that sulfated sites instead of phosphorylation sites were determined in our study.…”

Positive Mode LC-MS/MS Analysis of Chondroitin Sulfate Modified Glycopeptides Derived from Light and Heavy Chains of The Human Inter-α-Trypsin Inhibitor Complex*

“…Further, it has been demonstrated that sulfopeptides complexed to sodium ions were protected from fragmentation using positive mode electron capture/transfer dissociation (ECD/ETD) (45). Here, we explored if sodium ion complexation could be a feasible way of enhancing the stability of sulfate glycan substituents in positive mode MS/MS.…”

Metastable Atom-Activated Dissociation Mass Spectrometry of Phosphorylated and Sulfonated Peptides in Negative Ion Mode

“…All of the following peptides were provided by Protea Biosciences Inc. (Morgantown, WV, USA): the phosphorylated and non-phosphorylated forms of angiotensin II, cholecystokin (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and calcitonin (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29); the sulfated and non-sulfated forms of cholecystokinin (26)(27)(28)(29)(30)(31)(32)(33), leuenkephalin, and hirudin. Methanol (HPLC grade), ammonium hydroxide, and glacial acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).…”

“…To achieve improved ionization efficiency and modification retainment of highly acidic sulfonated peptides in ECD, charge enrichment through the formation of a divalent metal cation-peptide complex is necessary. In a similar experiment, Medzihradszky et al investigated the labile nature of sulfonated peptides that contained only one basic amino acid employing both ETD and ECD [23]. Under native ETD and ECD conditions, 15% (in the best case) of the -SO 3 modification retainment was observed for the multiply protonated cations [23].…”

“…In a similar experiment, Medzihradszky et al investigated the labile nature of sulfonated peptides that contained only one basic amino acid employing both ETD and ECD [23]. Under native ETD and ECD conditions, 15% (in the best case) of the -SO 3 modification retainment was observed for the multiply protonated cations [23]. The modification retainment improved to 73% (in the best case) when the peptides were complexed with Na + .…”