Investigation of ion/molecule reactions as a quantification method for phosphorylated positional isomers: An FT-ICR approach

Abstract

A rapid and accurate method of quantifying positional isomeric mixtures of phosphorylated hexose and N-acetylhexosamine monosacchrides by using gas-phase ion/molecule reactions coupled with FT-ICR mass spectrometry is described. Trimethyl borate, the reagent gas, reacts readily with the singly charged negative ions of phosphorylated monosaccharides to form two stable product ions corresponding to the loss of one or two neutral molecules of methanol from the original adduct. Product distribution in the ion/molecule reaction spectra differs significantly for isomers phosphorylated in either the 1-or the 6-position. As a result, the percents of total ion current of these product ions for a mixture of the two isomers vary with its composition. In order to determine the percentage of each isomer in an unknown mixture, a multicomponent quantification method is utilized in which the percents of total ion current of the two product ions for each pure monosaccharide phosphate and the mixture are used in a two-equation, two-unknown system. The applicability of this method is demonstrated by successfully quantifying mock mixtures of four different isomeric pairs: Glucose-1-phosphate and glucose-6-phosphate; mannose-1-phosphate and mannose-6-phosphate; galactose-1-phosphate and galactose-6-phosphate; N-acetylglucosamine-1-phosphate and N-acetylglucosamine-6-phosphate. The effects of mixture concentrations and ion/molecule reaction conditions on the quantification are also discussed. Our results demonstrate that this assay is a fast, sensitive, and robust method to quantify isomeric mixtures of phosphorylated monosaccharides. . In particular, the phosphate linkage position of carbohydrates is important to regulate biological function. For example, the conversion between the 6-phosphate and the 1-phosphate isomers of several monosaccharides, which is regulated by the corresponding phosphate mutases, plays an important role in the glycosylation of glycoproteins [5,6]. Specifically, the deficiency of the enzyme phosphomannomutase, which catalyzes the conversion of mannose-6-phosphate to mannose-1-phosphate in the early step of biosynthesis of lipid-linked oligosaccharides, is responsible for a disease called CDG-1a [1], in which glycoproteins with truncated N-linked carbohydrates are formed.Despite their importance, analysis of phosphorylated carbohydrates by traditional methods has been problematic due to their structural variety and non-characteristic UV absorbance. As a result, traditional enzyme kinetic measurements of the phosphate mutases mentioned above require up to three coupling enzymes [7][8][9]. Recently, Turecek et al. [10] reported an affinity capture and elution/electrospray ionization mass spectrometry assay of phosphomannomutase and phosphomannose isomerase. In their method, the product isomer was subjected to another enzymatic reaction that altered its mass in order to be detected by mass spectrometry. Direct quantitative analysis of phosphorylated glucose has been reported using anion-exchange chromatography ...

Comparison of Functional Group Selective Ion–Molecule Reactions of Trimethyl Borate in Different Ion Trap Mass Spectrometers

“…Ion-molecule reactions can be fast, efficient, and highly specific, properties which often are paramount to molecular characterization experiments. Ion-molecule reactions have been used for isomer differentiation [11][12][13][14], functional group identification [15][16][17][18][19][20], and detailed structural elucidation [21][22][23].…”

A novel mass spectrometric method to distinguish isobaric monosaccharides that are phosphorylated or sulfated using ion-pairing reagents

“…( [2][3][4]. They are also involved in glycolysis processes in the extracellular matrix [7,8]. Sulfation effects protein recognition and helps facilitate clearance of proteins and metabolites from the body [9 -14].…”

“…Phosphorylation is linked to signal transduction, gene expression, cell cycle, cytoskeletal regulation, and apoptosis [1][2][3][4][5][6][7][8]. In particular, phosphorylated carbohydrates are integral components of nucleic acids [1,5], oligosaccharides [1,5], glycopeptides [2][3][4]6], and glycoproteins [2][3][4].…”

“…In particular, phosphorylated carbohydrates are integral components of nucleic acids [1,5], oligosaccharides [1,5], glycopeptides [2][3][4]6], and glycoproteins [2][3][4]. They are also involved in glycolysis processes in the extracellular matrix [7,8]. Sulfation effects protein recognition and helps facilitate clearance of proteins and metabolites from the body [9 -14].…”

Identification of the Phenol Functionality in Deprotonated Monomeric and Dimeric Lignin Degradation Products via Tandem Mass Spectrometry Based on Ion–Molecule Reactions with Diethylmethoxyborane

“…However, most studies on functional group-specific ion-molecule reactions have focused on protonated analytes. Only in a few cases were deprotonated analytes examined [24][25][26][27][28], and to the best of our knowledge, no reagents have been developed for the identification of the phenol functionality. In this study, diethyl methoxyborane (DEMB) is explored as a reagent for the identification of the phenol functionality in deprotonated ligninrelated analytes.…”