Congruent Strategies for Carbohydrate Sequencing. 1. Mining Structural Details by MSⁿ

Abstract

This report is the first in a series of three focused on establishing congruent strategies for carbohydrate sequencing. The reports are divided into (i) analytical considerations that account for all aspects of small oligomer structure by MS n disassembly, (ii) database support using an ion fragment library and associated tools for highthroughput analysis, and (iii) a concluding algorithm for defining oligosaccharide topology from MS n disassembly pathways. The analytical contribution of this first report explores the limits of structural detail exposed by ion trap mass spectrometry with samples prepared as methyl derivatives and analyzed as metal ion adducts. This data mining effort focuses on correlating the fragments of small oligomers to stereospecific glycan structures, an outcome attributed to a combination of metal ion adduction and analyte conformation. Facile glycosidic cleavage introduces a point of lability (pyranosyl-1-ene) that upon collisional activation initiates subsequent ring fragmentation. Product masses and ion intensities vary with interresidue linkage, branching position, and monomer stereochemistry. Excessive fragmentation is the property of small oligomers where collisional energy within a smaller number of oscillators dissipates through extensive fragmentation. The procedures discussed in this report are unified into a singular strategy using an ion trap mass spectrometer with the sensitivity expected for electron multiplier detection. Although a small set of structures have been discussed, the basic principles considered are fully congruent, with ample opportunities for expansion.The proliferation of reports attributing biological function to oligosaccaharide epitopes continues unabated. To fully appreciate their specific biological roles, an improved accounting of carbohydrate structural details needs greater scrutiny and more judicious reporting. Unfortunately, a comprehensive strategy for carbohydrate sequencing is lacking. Over the past decades, the absence of an integrated strategy to fully define a carbohydrate sequence has been given little attention due to overwhelming and direct functional lineage of nucleic acid and protein polymers. Even selective strategies to assign components of structure show little focus toward congruency. The reporting of partial sequences, assumed motifs, sequence arrays devoid of linkage and branching definition, and multiple structures enclosed in brackets are currently acceptable conclusions in published reports. The general acceptance of such reports diminishes the driving force for the development of improved and comprehensive methodologies.A sequence definition should provide all the components of structure necessary for reporting or synthesis. Structural components should include monomer identification, positions of interresidue linkage, and array topology, where a linkage definition includes anomericity, and array topologies describe linear and branching sequence information. When considering molecular glycosylation, sites of conjugatio...

A glycomics platform for the analysis of permethylated oligosaccharide alditols

“…The branching pattern of the oligosaccharides could be deduced from CID spectra containing fragments bearing a mass signature of Ϫ14 Da for each lost branch. In samples containing Hex and HexNAc residues, abundant 0,4 A, 1,4 A, 1,3 A, and 3,5 A cross-ring fragments were often observed, thus allowing the assignment of 1-2, 1-4, and 1-6-linkages [37,38]. Elimination at the C3 of HexNAc was also observed, and provided a chemical signature that could be used to assign C3 substitutions as has been previously reported [17,18].…”

“…Online derivatization coupled with LC/MS of permethylated oligosaccharide alditols has obvious advantages, and we are in the process of adapting online derivatization to the LC/MS platform. Reinhold et al have recently reported the complete assignment of oligosaccharide structures of permethylated glycans using a database library, FragLib, in conjunction with their Oligosaccharide Subtree Constraint Algorithm (OSCAR) [38,54,55]. This algorithm incorporates analyst-selected MS n ion fragmentation pathways for elucidation of oligosaccharide topology using a top-down sequencing strategy.…”

Carbohydrate Structural Isomers Analyzed by Sequential Mass Spectrometry

“…In these examples, we will use many of the concepts and tools previously described. 7,8,15,[26][27][28] All structures were derived de novo with input data from a single instrument.…”

“…A large body of work has shown that spectral comparisons of isomers at different stages of sequential molecular fragmentation (MS n ) [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] are clearly different, although in one case, reproducibility has been reported to be "vague". 25 In our experience, spectral reproducibility has been excellent, 26 and this very feature has provided an opportunity to build a searchable fragment library 27 and derive glycan structure with supporting tools. 28 It has been effectively argued that the transfer of energy by gas-phase collisions maybe too broad to define the subtle isomeric features within carbohydrate structures.…”

“…The principles involved have been previously considered 26 and complex biological applications recently reported. 15 The overall focus has been to better define MS n as a universal sequencing strategy consistent with the goals of automation and HTP analysis.…”