Analysis of sulfated peptides using positive electrospray ionization tandem mass spectrometry

Citations Over TimeTop 16% of 2001 papers

Abstract

Abstract Presented is a method for analyzing sulfated peptides, and differentiating the post‐translational modification (PTM) from its isobaric counterpart phosphorylation, using quadrupole time‐of‐flight (Qq/TOF) mass spectrometry (MS) and positive ion nanoelectrospray MS/MS. A set of commercially available sulfo‐ and phosphopeptide standards was analyzed via in‐source dissociation and MS/MS to generate fragmentation signatures that were used to characterize and differentiate the two modifications. All of the phosphorylated peptides retained their +80 Da modifications under collision‐induced decomposition (CID) conditions and peptide backbone fragmentation allowed for the site‐specific identification of the modification. In sharp contrast, sulfated peptides lost SO 3 from the precursor as the collision energy (CE) was increased until only the non‐sulfated form of the peptide was observed. The number of 80 Da losses indicated the number of sulfated sites. By continuing to ramp the CE further, it was possible to fragment the non‐sulfated peptides and obtain detailed sequence information. It was not possible to obtain site‐specific information on the location of the sulfate moieties using positive ion MS/MS as none of the original precursor ions were present at the time of peptide backbone fragmentation. This method was applied to the analysis of recombinant human B‐domain deleted factor VIII (BDDrFVIII), which has six well‐documented sulfation sites and several potential phosphorylation sites located in two of the sulfated regions of the protein. Seven peptides with single and multiple +80 Da modifications were isolated and analyzed for their respective PTMs. The fragmentation patterns obtained from the BDDrFVIII peptides were compared with those obtained for the standard peptides; and in all cases the peptides were sulfated. None of the potential phosphorylation sites were found to be occupied, and these results are consistent with the literature. Copyright © 2001 John Wiley & Sons, Ltd.

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