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Hydrogen bonds are the dominant motif for organizing the three-dimensional structures of biomolecules such as carbohydrates, nucleic acids, and proteins, and serve as templates for proton transfer reactions. Computations, gas-phase acidity measurements, and pKa determinations in dimethyl sulfoxide on a series of polyols indicate that multiple hydrogen bonds to a single charged center lead to greatly enhanced acidities. A new class of Brønsted acids, consequently, is proposed. [ABSTRACT FROM AUTHOR]

N-linked glycosylation is one of the most common protein PTMs, and the topological structure (monosaccharide composition and sequence as well as glycosidic linkages) of N-glycans is vital information to understand their biological functions and roles. Tandem mass spectrometry has been widely used for topological structure characterization of N-glycans, where comprehensive understanding of fragmentation pathways and characteristics of product ions are essential to achieve best interpretation of MS/MS data and highest confidence of identification. Here, we report our glycomic study of N-glycome of mouse brain as well as fragmentation pathway analysis of the identified N-glycans. With LC-MS/MS analysis at both the positive and negative ESI modes together with our recently developed N-glycan database search engine GlySeeker, 221 unique N-glycans with putative topological structures were identified with target-decoy searches and number of best hits of 1. Analysis of fragmentation pathways and characteristics of product ions of permethylated N-glycans in the positive mode and native N-glycans in the negative mode were further carried out. The reported N-glycans serve as a basic reference for future glycosylation study of mouse brain; and in general database search of tandem mass spectra of N-glycans, B/Y/Z ions should be preferentially considered for the permethylated form in the positive mode and B/C/Z ions for the native form in the negative mode. [ABSTRACT FROM AUTHOR]

The soluble N-glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) can establish an N-glycosidic bond at the asparagine residue in the Asn-Xaa-Ser/Thr consensus sequon and is one of the most promising tools for N-glycoprotein production. Here, by integrating computational and experimental strategies, we revealed the molecular mechanism of the substrate recognition and following catalysis of ApNGT. These findings allowed us to pinpoint a key structural motif ( 215 DVYM 218 ) in ApNGT responsible for the peptide substrate recognition. Moreover, Y222 and H371 of ApNGT were found to participate in activating the acceptor Asn. The constructed models were supported by further crystallographic studies and the functional roles of the identified residues were validated by measuring the glycosylation activity of various mutants against a library of synthetic peptides. Intriguingly, with particular mutants, site-selective N-glycosylation of canonical or noncanonical sequons within natural polypeptides from the SARS-CoV-2 spike protein could be achieved, which were used to investigate the biological roles of the N-glycosylation in membrane fusion during virus entry. Our study thus provides in-depth molecular mechanisms underlying the substrate recognition and catalysis for ApNGT, leading to the synthesis of previously unknown chemically defined N-glycoproteins for exploring the biological importance of the N-glycosylation at a specific site., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Protein structural and functional studies rely on complete qualitative and quantitative information on protein species (proteoforms); thus, it is important to quantify differentially expressed proteins at their molecular level. Here we report our development of universal pseudoisobaric dimethyl labeling (pIDL) of amino groups at both the N-terminal and lysine residues for relative quantitation of intact proteins. Initial proof-of-principle study was conducted on standard protein myoglobin and hepatocellular proteomes (HepG2 vs LO2). The amino groups from both the N-terminal and lysine were dimethylated with HXHO (X = (13)C or C) and NaBY3CN (Y = H or D). At the standard protein level, labeling efficiency, effect of product ion size, and mass resolution on quantitation accuracy were explored; and a good linear quantitation dynamic range up to 50-fold was obtained. For the hepatocellular proteome samples, 33 proteins were quantified with RSD ≤ 10% from one-dimensional reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) analysis of the 1:1 mixed samples. The method in this study can be extended to quantitation of other intact proteome systems. The universal "one-pot" dimethyl labeling of all the amino groups in a protein without the need of preblocking of those on the lysine residues is made possible by protein identification and quantitation analysis using ProteinGoggle 2.0 with customized databases of both precursor and product ions containing heavy isotopes.

The ionization energy (IE) of the 3-cyclopropenyl radical (6.00 ± 0.17 eV) was measured in the gas phase by reacting 3-cyclopropenium cation (c-C3H3(+)) with a series of reference reagents of known IEs. This result was combined in a thermodynamic cycle to obtain the heat of formation of c-C3H3(•) (118.9 ± 4.0 kcal mol(-1)) and the allylic C-H bond dissociation energy (BDE) of cyclopropene (104.4 ± 4.0 kcal mol(-1)). These experimental values are well reproduced by high level G3 and W1 computations and reveal that the BDE is similar to that for cyclopropane and the vinyl position of cyclopropene. This is unprecedented and is a reflection of the unusual nature of cyclopropene.

Deprotonation of tyrosine in the gas phase was found to occur preferentially at the phenolic site, and the conjugate base consists of a 70:30 mixture of phenoxide and carboxylate anions at equilibrium. This result was established by developing a chemical probe for differentiating these two isomers, and the presence of both ions was confirmed by photoelectron spectroscopy. Equilibrium acidity measurements on tyrosine indicated that deltaG(acid)(o) = 332.5 +/- 1.5 kcal mol(-1) and deltaH(acid)(o) = 340.7 +/- 1.5 kcal mol(-1). Photoelectron spectra yielded adiabatic electron detachment energies of 2.70 +/- 0.05 and 3.55 +/- 0.10 eV for the phenoxide and carboxylate anions, respectively. The H/D exchange behavior of deprotonated tyrosine was examined using three different alcohols (CF3CH2OD, C6H5CH2OD, and CH3CH2OD), and incorporation of up to three deuterium atoms was observed. Two pathways are proposed to account for these results, and all of the experimental findings are supplemented with B3LYP/aug-cc-pVDZ and G3B3 calculations. In addition, it was found that electrospray ionization of tyrosine from a 3:1 (v/v) CH3OH/H2O solution using a commercial source produces a deprotonated [M-H]- anion with the gas-phase equilibrium composition rather than the structure of the ion that exists in aqueous media. Electrospray ionization from acetonitrile, however, leads largely to the liquid-phase (carboxylate) structure. A control molecule, p-hydroxybenzoic acid, was found to behave in a similar manner. Thus, the electrospray conditions that are employed for the analysis of a compound can alter the isomeric composition of the resulting anion.

Electrospray ionization of tyrosine from a 3:1 (v:v) CH3OH/H2O solution is found to afford an M - H ion which is a 70:30 mixture of phenoxide and carboxylate ions. This corresponds to the gas-phase equilibrium composition and not the liquid-phase proportions. In contrast, the carboxylate is produced as the dominant ion (approximately 95%) from anhydrous CH3CN and CH3CN/H2O mixtures. The addition of small amounts of CH3OH to the solvent, however, convert the M - H ion back into the gas-phase isomeric ratio. The isomeric structure therefore depends on the solvent system from which an ion is sprayed.

Penam acids 6-8 [i.e., (2S,5R,6R)-, (2S,5S,6R)-, and (2S,5R,6S)-isomers of 6-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid] were prepared by deesterification of the corresponding methyl esters 2-4. The same methodology applied to ester 1 did not lead to the (2S,5S,6S)-isomer 5 but rather a 72% yield of the thiazoline derivative 9. High-resolution mass spectrometry analysis of the reaction headspace gases indicated that a stoichiometric amount of carbon monoxide is produced during the deesterification of 1. A mechanism for this decarbonylation reaction is proposed. This appears to represent a new type of fragmentation reaction for a penam carboxylic acid. The free energies of various reaction species along viable decarbonylation reaction coordinates for acids 5 and 7 were computed by using the density functional theory method IEFPCM/M06/6-31+G(d). Anionic and zwitterionic (neutral) variants of the proposed mechanism were considered, but each produced computed activation free energies deemed to be too high (>45 kcal/mol) to be experimentally relevant. The computed activation free energies for the protonated (cationic) variant of the mechanism were 17.3 kcal/mol for 7 vs 8.8 kcal/mol for 5. The value of this difference in energies of activation (DeltaDeltaG++) is quite consistent with experimental observations and supports the proposed mechanism. For a portion of the computed reaction coordinate that involves ring opening of the lactam ring by an internal carboxylic acid group to form a cyclic anhydride, the expected tetrahedral intermediate was circumvented by a direct (concerted) and facile N- to O-acyl migration event. Additional thermal gas-phase reaction products produced during gas chromatographic analysis of the penams 1-8 were characterized with high-resolution mass spectrometry, and possible mechanisms for their formation are presented.

Hydrogen-deuterium exchange reactions of deprotonated amino acids and small peptides were studied. Selective labeling can be carried out at the alpha-amino group of lysine (2 of 4 labile hydrogens undergo exchange with CF3CH2OD) and the guanidine side chain of arginine (3 of 6 labile hydrogens undergo exchange with CH3CH2OD and C6H5CH2OD). Differential labeling of peptides also was accomplished, and the extent of H/D exchange is dependent on the amino acids which are present as well as their order in the chain.

Hydrogen-deuterium exchange experiments were carried out on the conjugate base of cysteine with four different deuterated alcohols. Three H/D exchanges are observed to take place in each case, and a relay mechanism which requires the SH and CO2H groups to have similar acidities and subsequently proceeds through a zwitterionic intermediate is proposed. Gas-phase acidity measurements also were carried out in a quadrupole ion trap using the extended kinetic method and in a Fourier transform mass spectrometer by an equilibrium determination. The results are in excellent accord with each other and high-level ab initio and density functional theory calculations and indicate that the side-chain thiol in cysteine is more acidic than the carboxyl group by 3.1 kcal mol-1. Deprotonated cysteine is thus predicted to be a thiolate ion. A zwitterionic species also was located on the potential energy surface, but it is energetically unfavorable (+10.1 kcal mol-1).

Both C-H bond dissociation energies for cyclobutene were measured in the gas phase (BDE = 91.2 +/- 2.3 (allyl) and 112.5 +/- 2.5 (vinyl) kcal mol-1) via a thermodynamic cycle by carrying out proton affinity and electron-binding energy measurements on 1- and 3-cyclobutenyl anions. The results were compared to those for an acyclic model compound, cis-2-butene, and provide the needed information to experimentally establish the heat of formation of cyclobutadiene. Chemically accurate G3 and W1 calculations also were carried out on cycloalkanes, cycloalkenes, and selected reference compounds. It appears that commonly cited bond energies for cyclopropane, cyclobutane, and cyclohexane are 3 to 4 kcal mol-1 too small and their pi bond strengths, as given by BDE1 - BDE2, are in error by up to 8 kcal mol-1.