Evaluation of low energy CID and ECD fragmentation behavior of mono-oxidized thio-ether bonds in peptides

Abstract

Thio-ether bonds in the cysteinyl side chain of peptides, formed with the most commonly used cysteine blocking reagent iodoacetamide, after conversion to sulfoxide, releases a neutral fragment mass in a low-energy MS/MS experiment in the gas phase of the mass spectrometer [6]. In this study, we show that the neutral loss fragments produced from the mono-oxidized thio-ether bonds (sulfoxide) in peptides, formed by alkyl halide or double-bond containing cysteine blocking reagents are different under low-energy MS/MS conditions. We have evaluated the low-energy fragmentation patterns of mono-oxidized modified peptides with different cysteine blocking reagents, such as iodoacetamide, 3-maleimidopropionic acid, and 4-vinylpyridine using FTICR-MS. We propose that the mechanisms of gas-phase fragmentation of mono-oxidized thio-ether bonds in the side chain of peptides, formed by iodoacetamide and double-bond containing cysteine blocking reagents, maleimide and vinylpyridine, are different because of the availability of acidic ␤-hydrogens in these compounds. Moreover, we investigated the fragmentation characteristics of mono-oxidized thio-ether bonds within the peptide sequence to develop novel mass-spectrometry identifiable chemical cross-linkers. This methionine type of oxidized thio-ether bond within the peptide sequence did not show anticipated low-energy fragmentation. Electron capture dissociation (ECD) of the side chain thio-ether bond containing oxidized peptides was also studied. ECD spectra of the oxidized peptides showed a greater extent of peptide backbone cleavage, compared with CID spectra. This fragmentation information is critical to researchers for accurate data analysis of this undesired modification in proteomics research, as well as other methods that may utilize sulfoxide derivatives. . Before digestion, the disulfide bonds in the proteins are first cleaved with either DTT or ␤-mercaptoethanol, and their reformation must be blocked using different cysteine blocking reagents. Several cysteine-blocking reagents exist; the most common are iodoacetamide, N-ethylmaleimide, acrylamide, and 4-vinylpyridine [2][3][4]. These methods that use iodoacetamide rely on an alkyl halide to react with the sulfhydryl group of cysteine. The other three blocking reagents, maleimide, acrylamide, and vinylpyridine utilize double bonds to react with the sulfhydryl group of cysteine by the Michael addition type mechanism [5]. All of these blocking reagents form thio-ether bonds in the cysteinyl side chain of proteins. Steen et al. reported that these thio-ether bonds can be oxidized in current protein profiling techniques, such as tryptic digestion and gel electrophoresis [6]. The oxidized thio-ether bond formed with iodoacetamide in the cysteinyl side chain of the peptide loses a neutral fragment mass in the gas phase under low-energy MS/MS conditions. Steen et al. exploited this low-energy fragmentation characteristic of mono-oxidized thio-ethers to profile phosphorylation by ␤-elimination and Michael addition chemical st...

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

“…H/D exchange experiments also confirmed that the neutral loss of CH 2 CHCONH 2 (CH 2 CHX=, where X= ϭ CONH 2 ) from these peptide ions occurred via a charge-remote cis-1,2 elimination reaction to yield the cysteine sulfenic acid-containing product ion (2), as shown in pathway B of Scheme 1. This is consistent with the mechanism recently proposed by Chowdhury et al for the neutral loss of 4-vinylpyridine from S-pyridylethyl cysteine sulfoxidecontaining°peptides° [8].…”

“…Consistent with these observations, the mechanism responsible for the loss of XSOH from S-alkyl cysteine sulfoxide-containing peptides formed by reaction with iodoacetamide, where X ϭ CH 2 CONH 2 , has been proposed to occur via a "charge-remote" cis-1,2 elimination reaction involving transfer of the ␣-hydrogen atom to yield an acyclic dehydroalanine-containing product ion (1) (Scheme 1, pathway A) [6,7]. A similar cis-1,2 elimination mechanism, yielding a cysteine sulfenic acid-containing product ion (2) (Scheme 1, pathway B), has also been proposed for the loss of CH 2 CHX= from S-alkyl cysteine sulfoxide-containing peptides formed by reaction with acrylamide (X= ϭ CONH 2 ) [5] or vinylpyridine (X= ϭ C 5 H 4 N) [8], as well as for the loss of C 6 H 7 NO 2 from S-alkyl cysteine sulfoxide-containing peptides formed by reaction with N-ethylmaleimide [8]. These mechanistic proposals are also consistent with those previously determined for the fragmentation of methionine sulfoxide-containing peptide ions, where the dominant side-chain loss of CH 3 SOH occurs via a charge-remote cis-1,2 elimination reaction under conditions of low proton mobility [14,15].…”

“…(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].…”

“…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 current database search algorithms [10,11] employed for the identification and characterization of these modified peptide ions.…”

“…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].…”