Study of the gas-phase fragmentation behaviour of sulfonated peptides

Abstract

a b s t r a c tA series of singly and doubly protonated peptides bearing sulfonated residue have been studied, using both experiment and molecular modelling, to elucidate fragmentation chemistry of sulfonated peptides. Collision-induced dissociation mass spectra indicate that the sulfo group loss (neutral loss of 80 Da) is the dominant dissociation channel. Modelling results suggest the proton transfer mechanism, where upon vibrational excitation, the acidic side chain proton is transferred from the sulfo group hydroxyl to the ester oxygen resulting in S O bond cleavage and formation of the unmodified hydroxyl containing residue and SO 3 . Conformations associated with potential energy profile of the reaction imply the charge remote nature of the proposed mechanism. The proposed proton transfer mechanism was compared with the intramolecular nucleophilic substitution (S N 2) mechanism, the main pathway suggested for neutral loss of phosphoric acid from phosphopeptides. Both pathways (proton transfer and S N 2) are available for sulfonated and phosphorylated peptides; however, each posttranslational modification favours different mechanism. The change of the bond dissociation enthalpies and the ability of stabilising the transition state structures are demonstrated as main factors responsible for each posttranslational modification activating a different pathway.

Development of a Sample-Preparation Workflow for Sulfopeptide Enrichment: From Target Analysis to Challenges in Shotgun Sulfoproteomics

“…Fifty-seven peptides were identified as phosphorylated, and 4 were identified as tyrosine-sulfated, respectively belonging to 46 and 3 protein groups (Table S2). MaxQuant searches tyrosine sulfopeptides by the 79.9568 mass shift in the precursor and the typical SO 3 neutral loss, as described in the literature for CID fragmentation of sulfopeptides in the positive-ionization mode, which is dominated by the neutral loss of SO 3 (79.956817) due to the proton-transfer mechanism being preferred over the S N 2 dissociation mechanism, which is, on the contrary, the main dissociation path for phosphopeptides and produces the H 3 PO 4 (97.976898) neutral loss. As such, if peptides with a given sequence modified with either sulfation or phosphorylation are nearly isobaric in the MS scan (difference between the −H 2 PO 3 and −HSO 3 moieties is only 9.6 mDa), the fragmentation mechanism in the MS/MS mode is different and can distinguish phosphorylation from sulfation, although site location cannot be afforded …”

“…The same issue was not observed for MaxQuant identifications. Spectra were manually checked for SO 3 neutral losses in the HCD spectrum . Some product ions matched by Proteome Discoverer were assigned as precursors, y- and b-ions also bearing the sulfate moiety, but no product obtained by further loss of SO 3 was found.…”

“…The differential analysis of sulfation and phosphorylation by MS is further complicated by the nearly isobaric nature of these modifications, as the difference between the −H 2 PO 3 and −HSO 3 moieties is only 9.6 mDa . To tackle this last issue, efforts have been made to find fragmentation strategies that could differentiate phosphate from sulfate, exploiting covalent modification (i.e., free-radical-initiated peptide sequencing based on (2,2,6,6-tetramethylpiperidin-1-yl)­oxyl (TEMPO) radical and negative-ion MS) or direct analysis under conventional positive-ionization mode (with noncovalent derivatization and ionization enhancer by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, collision-induced dissociation (CID), electron-capture dissociation (ECD), and electron-transfer dissociation (ETD)) or, more frequently, under negative-ionization mode (using UV photodissociation MS, negative-ion electron-capture dissociation, CID, ion/ion charge inversion/attachment with dipolar direct current (dc) collisional activation, and hydrogen attachment/abstraction dissociation) or both polarities . Recently, the ultrahigh resolution of an Orbitrap Fusion Lumos mass spectrometer was also tested to distinguish phospho- from sulfopeptides .…”

The Key Role of Metal Adducts in the Differentiation of Phosphopeptide from Sulfopeptide Sequences by High-Resolution Mass Spectrometry

“… 12 Covalent modification strategies were used to distinguish the two modifications. 13 As for direct analysis, several fragmentation strategies were investigated over the years, including collision-induced dissociation (CID), 14 , 15 electron-capture dissociation (ECD), electron-transfer dissociation (ETD), 16 UV photodissociation, 17 negative-ion ECD, 18 ion/ion charge inversion/attachment with dipolar direct current collisional activation, 19 and hydrogen attachment/abstraction dissociation. 20 Recently, ultrahigh-resolution MS by new generation instrumentation was suggested as a possible solution to this issue in the full scan acquisition mode, while ETD and electron-transfer/collision-induced dissociation (ETciD) could provide information on site localization and the sulfopeptide sequence.…”

Free Radical Initiated Peptide Sequencing for Direct Site Localization of Sulfation and Phosphorylation with Negative Ion Mode Mass Spectrometry

“…The elimination of a sulfate group in limited proton mobility environments has been calculated to require between 150 and 240 kcal mol -1 . 35,36 The preferential dissociation of the o -TEMPO-Bz moiety in the presence of these PTMs suggests that the homo-lytic cleavage must require lower energies than elimination of either PTM and, indeed, we calculate its dissociation energy to be 49.3 kcal mol −1 with B3LYP level theory using a 6–31G* basis set.…”