Oxonium Ion Guided Analysis of Quantitative Proteomics Data Reveals Site-Specific O-Glycosylation of Anterior Gradient Protein 2 (AGR2)

Developments in mass spectrometry (MS)-based analyses of glycoproteins have been important to study changes in glycosylation related to disease. Recently, the characteristic pattern of oxonium ions in glycopeptide fragmentation spectra had been used to assign different sets of glycopeptides. In particular, this was helpful to discriminate between O-GalNAc and O-GlcNAc. Here, we thought to investigate how such information can be used to examine quantitative proteomics data. For this purpose, we used tandem mass tag (TMT)-labeled samples from total cell lysates and secreted proteins from three different colorectal cancer cell lines. Following automated glycopeptide assignment (Byonic) and evaluation of the presence and relative intensity of oxonium ions, we observed that, in particular, the ratio of the ions at m/z 144.066 and 138.055, respectively, could be used to discriminate between O-GlcNAcylated and O-GalNAcylated peptides, with concomitant relative quantification between the different cell lines. Among the O-GalNAcylated proteins, we also observed anterior gradient protein 2 (AGR2), a protein which glycosylation site and status was hitherto not well documented. Using a combination of multiple fragmentation methods, we then not only assigned the site of modification, but also showed different glycosylation between intracellular (ER-resident) and secreted AGR2. Overall, our study shows the potential of broad application of the use of the relative intensities of oxonium ions for the confident assignment of glycopeptides, even in complex proteomics datasets.

Characterisation of hydration water in Nafion membrane

Hydration of fully dried Nafion membrane results in the formation of oxonium ions of increasing complexity, up to H9O4+. Beyond this, water behaves as the bulk liquid.

Experimental and computational evidence for C=O π-bonding in [CH2OH]+ and related oxonium ions

The question of whether [CH2OH]+ should be described as the hydroxymethyl cation, +CH2OH, or protonated formaldehyde, CH2=OH+, is reconsidered in the light of experimental information and new computational evidence. Previous arguments that the charge distribution in [CH2OH]+ may be probed by considering the incremental stabilisation of [CH2OH]+ induced by homologation on carbon (to give [CH3CHOH]+) or oxygen (to produce [CH2OCH3]+) are critically examined. Cation stabilisation energies are shown to be better indicators of the nature of these oxonium ions. Further insight into the structure of larger CnH2n+1O+ oxonium ions is obtained by considering the site of protonation of enol ethers and related species. Computational information, including AIM (Atoms and Molecules) and NBA (Natural Bond Analysis) charges on the carbon and oxygen atoms in [CH2OH]+ and related species, is considered critically. Particular attention is focused on the calculated bond lengths and barriers to rotation about the C–O bond(s) in [CH2OH]+, [CH3CHOH]+, [(CH3)2COH]+, CH3OH and [CH2OCH3]+ and the C–N bond in [CH2NH2]+. Trends in these data are consistent with appreciable π-bonding only in the C–O connections which correspond to the C=O bond in the parent aldehyde or ketone from which the oxonium ion may be considered to be derived by protonation or alkyl cationation.

Identifying Sialylation Linkages at the Glycopeptide Level by Glycosyltransferase Labeling Assisted Mass Spectrometry (GLAMS)

ABSTRACTPrecise assignment of sialylation linkages at the glycopeptide level is of importance in bottom-up glycoproteomics, and is also an indispensable step to understand the function of glycoproteins in pathogen-host interactions and cancer progression. Even though some efforts have been dedicated to the discrimination of α2,3/α2,6-sialylated isomers, unambiguous identification of sialoglycopeptide isomers is still needed. Herein, an innovative strategy of glycosyltransferase labeling assisted mass spectrometry (GLAMS) was developed. After specific enzymatic labeling, oxonium ions from higher-energy C-trap dissociation (HCD) fragmentation of α2,3-sailoglycopeptides generate unique reporters to distinctly differentiate those of α2,6-sailoglycopeptide isomers. Using this strategy, a total of 1,236 linkage-specific sialoglycopeptides were successfully identified from 161 glycoproteins in human serum.Abstract Figure

DIALib: an automated ion library generator for data independent acquisition mass spectrometry analysis of peptides and glycopeptides

Data Independent Acquisition (DIA) Mass Spectrometry (MS) workflows allow unbiased measurement of all detectable peptides from complex proteomes, but require ion libraries for interrogation of peptides of interest. These DIA ion libraries can be theoretical or built from peptide identification data from Data Dependent Acquisition (DDA) MS workflows. However, DDA libraries derived from empirical data rely on confident peptide identification, which can be challenging for peptides carrying complex post-translational modifications. Here, we present DIALib, software to automate the construction of peptide and glycopeptide Data Independent Acquisition ion Libraries. We show that DIALib theoretical ion libraries can identify and measure diverse N- and O-glycopeptides from yeast and mammalian glycoproteins without prior knowledge of the glycan structures present. We present proof-of-principle data from a moderately complex yeast cell wall glycoproteome and a simple mixture of mammalian glycoproteins. We also show that DIALib libraries consisting only of glycan oxonium ions can quickly and easily provide a global compositional glycosylation profile of the detectable “oxoniome” of glycoproteomes. DIALib will help enable DIA glycoproteomics as a complementary analytical approach to DDA glycoproteomics.

Capturing Nature's Oxonium Ions

<p><a></a>Acetylcholine and <i>S</i>-adenosylmethionine exemplify the tetraalkylammonium (R₄N⁺) and trialkylsulfonium (R₃S⁺) ions used by Nature. The corresponding trialkyloxonium ions (R₃O⁺), however, do not play a central role in biology most likely due to their hydrolytic instability compared with their ammonium and sulfonium counterparts. Indeed, Meerwein’s salts [(CH₃)₃O⁺BF₄^– and (CH₃CH₂)₃O⁺BF₄^–], the simplest of the trialkyloxonium ions, are among the most powerful alkylating agents known, and they too are unstable to water. Only recently have water stable trialkyloxonium ions been reported which contain an oxatriquinane skeleton. Interestingly, despite the inherent hydrolytic instability of the vast majority of trialkyloxonium ions, they have been postulated as key intermediates in the biosynthesis of a number of complex natural products from <i>Laurencia</i> species. The existence of these complex trialkyloxonium ions has been implied from the structural and stereochemical diversity of these natural products and is supported by elegant biomimetic total syntheses, yet no direct evidence for their existence has been forthcoming. Herein, we report the synthesis and full characterisation of one family of these biosynthetically relevant trialkyloxonium ions - the most structurally and stereochemically complex oxonium ions characterised to date. Additionally, the elucidation of their <i>in vitro </i>reactivity profile has resulted in the synthesis of more than ten complex halogenated natural products. This work substantiates the existence of complex trialkyloxonium ions as key reactive intermediates in the biosynthesis of numerous halogenated natural products from <i>L. </i>spp. – expanding Nature’s rich inventory of onium ions.</p>

Capturing Nature's Oxonium Ions

<p><a></a>Acetylcholine and <i>S</i>-adenosylmethionine exemplify the tetraalkylammonium (R₄N⁺) and trialkylsulfonium (R₃S⁺) ions used by Nature. The corresponding trialkyloxonium ions (R₃O⁺), however, do not play a central role in biology most likely due to their hydrolytic instability compared with their ammonium and sulfonium counterparts. Indeed, Meerwein’s salts [(CH₃)₃O⁺BF₄^– and (CH₃CH₂)₃O⁺BF₄^–], the simplest of the trialkyloxonium ions, are among the most powerful alkylating agents known, and they too are unstable to water. Only recently have water stable trialkyloxonium ions been reported which contain an oxatriquinane skeleton. Interestingly, despite the inherent hydrolytic instability of the vast majority of trialkyloxonium ions, they have been postulated as key intermediates in the biosynthesis of a number of complex natural products from <i>Laurencia</i> species. The existence of these complex trialkyloxonium ions has been implied from the structural and stereochemical diversity of these natural products and is supported by elegant biomimetic total syntheses, yet no direct evidence for their existence has been forthcoming. Herein, we report the synthesis and full characterisation of one family of these biosynthetically relevant trialkyloxonium ions - the most structurally and stereochemically complex oxonium ions characterised to date. Additionally, the elucidation of their <i>in vitro </i>reactivity profile has resulted in the synthesis of more than ten complex halogenated natural products. This work substantiates the existence of complex trialkyloxonium ions as key reactive intermediates in the biosynthesis of numerous halogenated natural products from <i>L. </i>spp. – expanding Nature’s rich inventory of onium ions.</p>

Capturing Nature's Oxonium Ions

<p><a></a>Acetylcholine and <i>S</i>-adenosylmethionine exemplify the tetraalkylammonium (R₄N⁺) and trialkylsulfonium (R₃S⁺) ions used by Nature. The corresponding trialkyloxonium ions (R₃O⁺), however, do not play a central role in biology most likely due to their hydrolytic instability compared with their ammonium and sulfonium counterparts. Indeed, Meerwein’s salts [(CH₃)₃O⁺BF₄^– and (CH₃CH₂)₃O⁺BF₄^–], the simplest of the trialkyloxonium ions, are among the most powerful alkylating agents known, and they too are unstable to water. Only recently have water stable trialkyloxonium ions been reported which contain an oxatriquinane skeleton. Interestingly, despite the inherent hydrolytic instability of the vast majority of trialkyloxonium ions, they have been postulated as key intermediates in the biosynthesis of a number of complex natural products from <i>Laurencia</i> species. The existence of these complex trialkyloxonium ions has been implied from the structural and stereochemical diversity of these natural products and is supported by elegant biomimetic total syntheses, yet no direct evidence for their existence has been forthcoming. Herein, we report the synthesis and full characterisation of one family of these biosynthetically relevant trialkyloxonium ions - the most structurally and stereochemically complex oxonium ions characterised to date. Additionally, the elucidation of their <i>in vitro </i>reactivity profile has resulted in the synthesis of more than ten complex halogenated natural products. This work substantiates the existence of complex trialkyloxonium ions as key reactive intermediates in the biosynthesis of numerous halogenated natural products from <i>L. </i>spp. – expanding Nature’s rich inventory of onium ions.</p>