Mechanisms for the selective gas-phase fragmentation reactions of methionine side chain fixed charge sulfonium ion containing peptides

The gas-phase fragmentation mechanisms of small models for peptides containing intermolecular disulfide links have been studied using a combination of tandem mass spectrometry experiments, isotopic labeling, structural labeling, accurate mass measurements of product ions, and theoretical calculations (at the MP2/6-311 ϩ G(2d,p)//B3LYP/3-21G(d) level of theory). Cystine and its C-terminal derivatives were observed to fragment via a range of pathways, including loss of neutral molecules, amide bond cleavage, and S-S and C-S bond cleavages. Various mechanisms were considered to rationalize S-S and C-S bond cleavage processes, including charge directed neighboring group processes and nonmobile proton salt bridge mechanism. Three low-energy fragmentation pathways were identified from theoretical calculations on cystine N-methyl amide: (1) S-S bond cleavage dominated by a neighboring group process involving the C-terminal amide N to form either a protonated cysteine derivative or protonated sulfenyl amide product ion (44.3 kcal mol Ϫ1 ); (2) C-S bond cleavage via a salt bridge mechanism, involving abstraction of the ␣-hydrogen by the N-terminal amino group to form a protonated thiocysteine derivative (35.0 kcal mol Ϫ1 ); and (3) C-S bond cleavage via a Grob-like fragmentation process in which the nucleophilic N-terminal amino group forms a protonated dithiazolidine (57.9 kcal mol Ϫ1 ). Interestingly, C-S bond cleavage by neighboring group processes have high activation barriers (63.1 kcal mol Ϫ1 ) and are thus not expected to be accessible during low-energy CID experiments. In comparison to the energetics of simple amide bond cleavage, these S-S and C-S bond cleavage reactions are higher in energy, which helps rationalize why bond cleavage processes involving the disulfide bond are rarely observed for low-energy CID of peptides with mobile proton(s) containing intermolecular disulfide bonds. On the other hand, the absence of a mobile proton appears to "switch on" disulfide bond cleavage reactions, which can be rationalized by the salt bridge mechanism. This potentially has important ramifications in explaining the prevalence of disulfide bond cleavage in singly protonated peptides under MALDI conditions. [6,7] in the MS/MS of protonated peptides are indicators of phosphorylation and methionine sulfoxide formation, respectively. Progress has been made in understanding the factors that govern these reactions through a combination of mechanistic studies [1,2,6,8] and interrogation of databases of tandem mass spectra [7]. Several different types of mechanisms may operate depending on the structure and properties of the peptide. For example, studies on simple derivatives of O-phosphoserine reveal that H 3 PO 4 loss can occur via the following: charge directed neighboring group process (path A of Scheme 1, X ϭ H 2 PO 4 ); and charge remote internal elimination reaction to form a dehydroalanine residue (path B of Scheme 1, X ϭ H 2 PO 4 ) [8]. These different pathways can

Section: When Will Disulfide Bond Cleavage Occur?mentioning

confidence: 99%

A novel salt bridge mechanism highlights the need for nonmobile proton conditions to promote disulfide bond cleavage in protonated peptides under low-energy collisional activation

Lioe

O’Hair

2007

“…CID-MS 3 of the neutral loss product ion from Figure 3a (see Figure 3b) gave rise to a range of sequence-type product ions resulting from cleavages along the peptide backbone, from which the peptide sequence and site of phosphorylation could potentially be derived, together with some relatively low abundance non-sequence-type ions corresponding to losses of the phosphate group and/or water from the precursor or sequence-type product ions. It is expected that as the proton affinity of the product ion structure formed via the loss of dimethylsulfide during CID-MS/MS (previously proposed to be a cyclic iminohydrofuran (IHF) moiety [58, 60]) will be higher than that of an amino group [60], the neutral loss product ion that is initially formed may have decreased proton mobility compared to that of the unmodified peptide of the same charge state. Decreased proton mobility within IHF-modified phosphopeptides could lead to more abundant loss of the phosphate group or increase the potential for phosphate group ‘scrambling’ under CID-MS 3 conditions [12], which therefore could limit the extent of unambiguous sequence information for subsequent phosphopeptide identification and characterization.…”

Section: Resultsmentioning

confidence: 99%

“…We have previously reported that peptides containing ‘fixed charge’ dialkylsulfonium ion derivatives located on selected functional groups of certain amino acids, including the thioether side chain of methionine (formed by reaction with phenacylbromide under acidic conditions) [56-58], the thiol side chain of cysteine (formed by reaction with 3-([ N -bromoacetamido]propyl)-methylphenacylsulfonium bromide (BAPMPS)) [59], or the amino side chain of lysine or peptide N-termini (formed by reaction with S,S ′-dimethylth-iobutanoylhydroxysuccinimide ester (DMBNHS)) [60] all undergo the exclusive neutral loss(es) of dialkylsulfide groups upon CID-MS/MS, independently of the amino acid composition and precursor ion charge state (i.e. ; proton mobility), thereby enabling the selective identification of derivatized peptides from within complex mixtures.…”

Section: Introductionmentioning

confidence: 99%

Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization

Zhou

Stemmer

et al. 2011

Self Cite

An amine specific peptide derivatization strategy involving the use of novel isobaric stable isotope encoded ‘fixed charge’ sulfonium ion reagents, coupled with an analysis strategy employing capillary HPLC, ESI-MS, and automated data dependent ion trap CID-MS/MS, -MS3, and/or ETD-MS/MS, has been developed for the improved quantitative analysis of protein phosphorylation, and for identification and characterization of their site(s) of modification. Derivatization of 50 synthetic phosphopeptides with S,S′-dimethylthiobutanoylhydroxysuccinimide ester iodide (DMBNHS), followed by analysis using capillary HPLC-ESI-MS, yielded an average 2.5-fold increase in ionization efficiencies and a significant increase in the presence and/or abundance of higher charge state precursor ions compared to the non-derivatized phosphopeptides. Notably, 44% of the phosphopeptides (22 of 50) in their underivatized states yielded precursor ions whose maximum charge states corresponded to +2, while only 8% (4 of 50) remained at this maximum charge state following DMBNHS derivatization. Quantitative analysis was achieved by measuring the abundances of the diagnostic product ions corresponding to the neutral losses of ‘light’ (S(CH3)2) and ‘heavy’ (S(CD3)2) dimethylsulfide exclusively formed upon CID-MS/MS of isobaric stable isotope labeled forms of the DMBNHS derivatized phosphopeptides. Under these conditions, the phosphate group stayed intact. Access for a greater number of peptides to provide enhanced phosphopeptide sequence identification and phosphorylation site characterization was achieved via automated data-dependent CID-MS3 or ETD-MS/MS analysis due to the formation of the higher charge state precursor ions. Importantly, improved sequence coverage was observed using ETD-MS/MS following introduction of the sulfonium ion fixed charge, but with no detrimental effects on ETD fragmentation efficiency.

“…Hydrogen/deuterium exchange can readily be used to differentiate between the charge-remote and charge-directed fragmentation pathways proposed in 5 , and y 6 ions in this spectrum, corresponding to cleavage of the amide bonds adjacent to the site of the cysteine side chain, suggests that the E2 elimination reaction had occurred. However, based on our recent study on the mechanisms responsible for the side-chain fragmentation reactions of methionine fixed charge sulfonium ion-containing peptides, where formation of a mixture of cyclic five-and six-membered product ions were°found°to°be°favored° [19],°the°S N 2 neighboring group participation reactions cannot be ruled out. In°contrast°to°the°data°shown°in°Figure°4a,°b,°and Table°1,°where°the°product°ions°formed°via°the°losses of H 2 NCOCH 2 CH 2 SOH and CH 2 CHCONH 2 (and/or CH 2 CHCONH 2 ϩH 2 O) from the S-amidoethyl cysteine sulfoxide-containing peptides were observed at similar abundances for all the precursor ion charge states examined (i.e., independently of the proton mobility of the peptide ions), the product ion abundances of the side-chain losses from S-pyridylethyl cysteine sulfoxide-containing peptides were observed to vary significantly depending on the proton mobility°of°the°precursor°ion.°For°example,°Figure°6°shows the product ion spectra obtained by CID-MS/MS of the singly, doubly, and triply protonated precursor ions of the S-pyridylethyl cysteine sulfoxide-containing peptide VTMGHFCNFGK [M(ox)C(S-pe) ( (Figure°6b°and°c,°respectively), were observed as the most abundant products.…”

Section: Multistage Tandem Mass Spectrometry H/d Exchange Reactionsmentioning

confidence: 99%

Mechanisms for the proton mobility-dependent gas-phase fragmentation reactions of S-alkyl cysteine sulfoxide-containing peptide ions

Froelich

Reid

2007

Self Cite

Mechanisms for the gas-phase fragmentation reactions of singly and multiply protonated precursor ions of the model S-alkyl cysteine sulfoxide-containing peptides GAILCGAILK, GAILCGAILR, and VTMGHFCNFGK prepared by reaction with iodomethane, iodoacetamide, iodoacetic acid, acrylamide, or 4-vinylpyridine, followed by oxidation with hydrogen peroxide, as well as peptides obtained from an S-carboxyamidomethylated and oxidized tryptic digest of bovine serum albumin, have been examined using multistage tandem mass spectrometry, hydrogen/deuterium exchange and molecular orbital calculations (at the B3LYP/6-31 ϩ G(d,p) level of theory). Consistent with previous reports, CID-MS/MS of the S-alkyl cysteine sulfoxide-containing peptide ions resulted in the dominant "non-sequence" neutral loss of an alkyl sulfenic acid (XSOH) from the modified cysteine side chains under conditions of low proton mobility, irrespective of the alkylating reagent employed. Dissociation of uniformly deuterated precursor ions of these model peptides determined that the loss of alkyl sulfenic acid in each case occurred via a "charge-remote" five-centered cis-1,2 elimination reaction to yield a dehydroalanine-containing product ion. Similarly, the charge state dependence to the mechanisms and product ion structures for the losses of CO 2 , CO 2 ϩ H 2 O and CO 2 ϩ CH 2 O from S-carboxymethyl cysteine sulfoxide-containing peptides, and for the losses of CH 2 CHCONH 2 and CH 2 CHC 5 H 4 N, respectively, from S-amidoethyl and S-pyridylethyl cysteine sulfoxide-containing peptide ions have also been determined. The results from these studies indicate that both the proton mobility of the peptide precursor ion and the nature of the S-alkyl substituent have a significant influence on the abundances and charge states of the product ions resulting from the various competing fragmentation pathways. (J Am Soc Mass Spectrom 2007, 18, 1690 -1705) © 2007 American Society for Mass Spectrometry C onventional approaches for mass spectrometry based protein identification and characterization typically involve the reduction and alkylation of cysteine residues before enzymatic digestion [1][2][3][4]. Numerous reports in the literature have demonstrated that the resulting thio-ether bonds are susceptible to oxidation, either during the enzymatic digestion process or subsequent sample handling steps, before mass spectrometry analysis [5][6][7][8]. These reports have also demonstrated that fragmentation of the resultant S-alkyl cysteine sulfoxide-containing peptides under lowenergy collision induced dissociation (CID)-tandem mass spectrometry (MS/MS) conditions can result in the formation of abundant product ions via cleavages occurring within the modified cysteine side chains [5][6][7][8]. While these "non-sequence" ions are indicative of the presence of the modified amino acid residue within the peptide, their formation at high abundance may "suppress" the formation of desired "sequence" ion information, thereby limiting the utility of "de novo" analysis strategies [9] or...