Richard A. J. O’Hair

MR1 presents microbial vitamin B metabolites to MAIT cells

Antigen-presenting molecules, encoded by the major histocompatibility complex (MHC) and CD1 family, bind peptide- and lipid-based antigens, respectively, for recognition by T cells. Mucosal-associated invariant T (MAIT) cells are an abundant population of innate-like T cells in humans that are activated by an antigen(s) bound to the MHC class I-like molecule MR1. Although the identity of MR1-restricted antigen(s) is unknown, it is present in numerous bacteria and yeast. Here we show that the structure and chemistry within the antigen-binding cleft of MR1 is distinct from the MHC and CD1 families. MR1 is ideally suited to bind ligands originating from vitamin metabolites. The structure of MR1 in complex with 6-formyl pterin, a folic acid (vitamin B9) metabolite, shows the pterin ring sequestered within MR1. Furthermore, we characterize related MR1-restricted vitamin derivatives, originating from the bacterial riboflavin (vitamin B2) biosynthetic pathway, which specifically and potently activate MAIT cells. Accordingly, we show that metabolites of vitamin B represent a class of antigen that are presented by MR1 for MAIT-cell immunosurveillance. As many vitamin biosynthetic pathways are unique to bacteria and yeast, our data suggest that MAIT cells use these metabolites to detect microbial infection.

A mass spectrometric and ab initio study of the pathways for dehydration of simple glycine and cysteine-containing peptide [M+H]+ ions

The gas phase fragmentation reactions of the [M+H]+ and [M+H−H2O]+ ions of glycylglycine, glycylcysteine, N-acetylglycine, N-acetylcysteine, their corresponding methyl esters, as well as several other related model systems have been examined by electrospray ionization (ESI) tandem mass spectrometry (MSn) using triple quadrupole and quadrupole ion trap mass spectrometers. Two discrete gas phase fragmentation pathways for the loss of water from glycine-containing peptides, corresponding to retro-Koch and retro-Ritter type reactions were observed. Two pathways were also observed for the loss of water from C-terminal cysteine-containing peptides: a retro-Koch type reaction and an intramolecular nucleophilic attack at the carbonyl of the amide bond by the cysteinyl side chain thiol. Various intermediates involved in these reactions, derived from the [M+H−H2O]+ ions of N-formylglycine and N-formylcysteine, were modeled using ab initio calculations at the MP2(FC)/6-31G*//HF/6-31G* level of theory. These calculations indicate that: (i) the retro-Koch reaction product is predicted to be more stable than the product from the retro-Ritter reaction for N-formylglycine, and (ii) the intramolecular nucleophilic attack product is preferred over the retro-Koch and retro-Ritter reaction products for N-formylcysteine. The results from these ab initio calculations are in good agreement with the experimentally determined ion abundances for these processes.

Do all b2 ions have oxazolone structures? Multistage mass spectrometry and ab initio studies on protonated N-acyl amino acid methyl ester model systems

Abstract The tandem mass spectrometry fragmentation reactions of 21 protonated N-acyl amino acid methyl esters are examined as models for more complicated peptides. Four main types of reactions are observed: loss of CH2CO from the N-terminal acetyl group; loss of CH3OH from the C-terminal ester group to yield a model system for a b2 ion structure; loss of water from amino acids without an OH side chain group; fragmentation of the side chain by way of small molecule loss (e.g. H2O, NH3, and CH3SH). CH3OH loss is the only common reaction observed for all systems. The resultant [M+H−CH3OH]+ ions were examined in further detail by way of MS3 experiments because previous studies have shown that the oxozolone structures liberate CO. Only lysine and arginine do not fragment by way of CO, which is suggestive of alternative cyclic structures involving the side chain. Ab initio calculations (at the MP2/6-31G∗//HF/6-31G∗) were carried out on isomeric b2 ions of both types (oxazolone and that involving side-chain interaction) derived from arginine, histidine, lysine, methionine, asparagine, glutamine, and serine. For arginine, histidine, and lysine the cyclic structures involving the side chain are more stable than the oxozolone structures. Finally, solution phase data relevant to the gas phase processes are highlighted.

GAS PHASE ION-MOLECULE REACTIONS IN A MODIFIED ION TRAP: H/D EXCHANGE OF NON-COVALENT COMPLEXES AND COORDINATIVELY UNSATURATED PLATINUM COMPLEXES

A commercially available electrospray ionization ion trap mass spectrometer has been modified to carry out gas phase ion–molecule reactions. The ability to study gas phase ion–molecule reactions in conjunction with collision induced dissociation (CID) based methods and the multistage trapping capabilities of the ion trap have been exploited in two ways: (i) gas phase H/D exchange reactions inside the ion trap, coupled with CID tandem mass spectrometry have been used to provide insights into the reactivity of non covalent complexes of amino acids and simple peptides, and (ii) CID prior to performing ion–molecule reactions has been used to synthesize and examine the reactivity of coordinatively unsaturated platinum complexes.

Electron capture dissociation and infrared multiphoton dissociation of oligodeoxynucleotide dications

We report electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) of doubly protonated and protonated/alkali metal ionized oligodeoxynucleotides. Mass spectra following ECD of the homodeoxynucleotides polydC, polydG, and polydA contain w or d “sequence” ions. For polydC and polydA, the observed fragments are even-electron ions, whereas radical w/d ions are observed for polydG. Base loss is seen for polydG and polydA but is a minor fragmentation pathway in ECD of polydC. We also observe fragment ions corresponding to w/d plus water in the spectra of polydC and d(GCATGC). Although the structure of these ions is not clear, they are suggested to proceed through a pentavalent phosphorane intermediate. The major fragment in ECD of d(GCATGC) is a d ion. Radical a- or z-type fragment ions are observed in most cases. IRMPD primarily results in base loss, but backbone fragmentation is also observed. IRMPD provides more sequence information than ECD, but the spectra are more complex due to extensive base and water losses. It is proposed that the smaller degree of sequence coverage in ECD, with fragmentation mostly occurring close to the ends of the molecules, is a consequence of a mechanism in which the electron is captured at a P=O bond, resulting in a negatively charged phosphate group. Consequently, at least two protons (or alkali metal cations) must be present to observe a w or d fragmention, a requirement that is less likely for small fragments.

Leaving group and gas phase neighboring group effects in the side chain losses from protonated serine and its derivatives.

The gas phase fragmentation reactions of protonated serine and its YNHCH(CH2X)CO2H derivatives, β-chloroalanine, S-methyl cysteine, O-methyl serine, and O-phosphoserine, as well as the corresponding N-acetyl model peptides have been examined via electrospray ionization tandem mass spectrometry (MS/MS). In particular, the competition between losses from the side chain and the combined loss of H2O and CO from the C-terminal carboxyl group of the amino acids or H2O or CH2CO from the N-acetyl model peptides are compared. In this manner the effect of the leaving group (Y = H or CH3CO, vary X) or of the neighboring group can be examined. It was found that the amount of HX lost from the side chain increases with the proton affinity of X [OP(O)(OH)2 > OCH3 ∼ OH > Cl]. The ion due to the side chain loss of H2O from the model peptide N-acetyl serine is more abundant than that from protonated serine, suggesting that the N-acetyl group is a better neighboring group than the amino group. Ab initio calculations at the MP2(FC)/6-31G*//HF/6-31G* level of theory suggest that this effect is due to the transition state barrier for water loss from protonated N-acetyl serine being lower than that for protonated serine. The mechanism for side chain loss has been examined using MS3 tandem mass spectrometry, independent synthesis of proposed product ion structures combined with MS/MS, and hydrogen/deuterium exchange. Neighboring group rather than cis 1,2 elimination processes dominate in all cases. In particular, the loss of H3PO4 from O-phosphoserine and N-acetyl O-phosphoserine is shown to yield a 3-membered aziridine ring and 5-membered oxazoline ring, respectively, and not the dehydroalanine moiety. This is in contrast to results presented by DeGnore and Qin (J. Am. Soc. Mass Spectrom.1998, 9, 1175–1188) for the loss of H3PO4 from larger peptides, where dehydroalanine was observed. Alternate mechanisms to cis 1,2 elimination, for the formation of dehydroalanine in larger phosphoserine or phosphothreonine containing peptides, are proposed.

Probing the fragmentation reactions of protonated glycine oligomers via multistage mass spectrometry and gas phase ion molecule hydrogen/deuterium exchange

Abstract An ion trap mass spectrometer equipped with electrospray ionization has been modified to study the structure of protonated polyglycyl peptides G n (where n = 2–5 glycine residues) and their product ions formed by collision induced dissociation tandem mass spectrometry (CID MS/MS) via the novel application of gas phase ion–molecule hydrogen/deuterium (H/D) exchange reactions. In particular, the structures of the b 2 , b 3 , b 4 , and b 5 ions formed via CID MS/MS from various protonated glycine oligomer precursors have been examined. The b 2 ions, formed from the protonated G 2 and G 3 precursor ions, the b 3 ion from the protonated G 3 precursor, and the b 4 ion from the protonated G 5 ion all undergo CID and gas phase H/D exchange consistent with formation of protonated oxazolone structures previously proposed for b n -type ions. However, CID MS/MS, MS 3 , and H/D exchange of the putative b 4 and b 5 arising from the protonated G 4 and G 5 precursor ions, respectively, as well as experiments with various methylated derivatives of G 4 , suggest that the major portion of these ions are not b n ions, but are instead formed via backbone–backbone neighboring group participation reactions remote to the C-terminal amino acid. Efforts to elucidate the mechanisms behind this loss of H 2 O are described.

Gas-Phase Fragmentation of Long-Lived Cysteine Radical Cations Formed Via NO Loss from Protonated S-Nitrosocysteine

In this work, we describe two different methods for generating protonated S-nitrosocysteine in the gas phase. The first method involves a gas-phase reaction of protonated cysteine with t-butylnitrite, while the second method uses a solution-based transnitrosylation reaction of cysteine with S-nitrosoglutathione followed by transfer of the resulting S-nitrosocysteine into the gas phase by electrospray ionization mass spectrometry (ESI-MS). Independent of the way it was formed, protonated S-nitrosocysteine readily fragments via bond homolysis to form a long-lived radical cation of cysteine (Cys•+), which fragments under collision-induced dissociation (CID) conditions via losses in the following relative abundance order: •COOH ≫ CH2S > •CH2SH-H2S. Deuterium labeling experiments were performed to study the mechanisms leading to these pathways. DFT calculations were also used to probe aspects of the fragmentation of protonated S-nitrosocysteine and the radical cation of cysteine. NO loss is found to be the lowest energy channel for the former ion, while the initially formed distonic Cys•+ with a sulfur radical site undergoes proton and/or H atom transfer reactions that precede the losses of CH2S, •COOH, •CH2SH, and H2S.

Side-chain involvement in the fragmentation reactions of the protonated methyl esters of histidine and its peptides

Abstract The gas phase fragmentation reactions of [M+H]+ ions of the methyl esters of histidine and histidine containing di- and tripeptides were examined by electrospray ionization (ESI) multistage mass spectrometry (MSn) experiments using a quadrupole ion trap mass spectrometer. The MS/MS spectra tend to be dominated by bn sequence ions, whose structures were probed via MS3 experiments and ab initio calculations at the MP2(FC)/6-31G∗//HF6-31G∗ level of theory (for bn ions where n = 1 and 2). The ab initio calculations suggest a structure for the b1 ion that is stabilized by the formation of a bicyclic ring via involvement of the side-chain imidazole ring. In contrast, MS3 experiments reveal that the b2 ion derived from the sequences HG-Y and GH-Y yield identical spectra to the MS/MS spectrum of the protonated diketopiperazine of (GH). These experimental results are consistent with ab initio calculations that reveal the side-chain protonated diketopiperazine of (GH) to be thermodynamically favored over all other b2 isomeric structures. Thus, the histidine side chain appears to exert both a direct and an indirect (through base catalysis) role in the formation of bn sequence ions from protonated peptides.

Involvement of salt bridges in a novel gas phase rearrangement of protonated arginine-containing dipeptides which precedes fragmentation

Abstract A novel gas phase rearrangement reaction has been discovered for [M+H] + ions of arginine-containing dipeptides. In the case of Gly-Arg and Arg-Gly, this leads to identical tandem mass spectra (MS/MS) thereby precluding their sequence assignment. Density functional theory (DFT) calculations and further multistage mass spectrometry experiments suggest a mechanism which involves the formation of salt bridges for Gly-Arg and Arg-Gly which then undergo ring closure followed by ring opening to form a mixed anhydride. Prevention of salt bridge formation switches off this reaction and yields different MS/MS spectra which allow sequence assignment. This can be achieved by preforming CID on the deprotonated dipeptides or their protonated methyl esters.