Congruent Strategies for Carbohydrate Sequencing. 2. FragLib:  An MSⁿ Spectral Library

Abstract

A bottom-up approach to achieve full oligosaccharide and glycan characterization has been described that is based on an MS n fragment spectral library and associated tools. The library, identified as FragLib, was initiated with known standards and commercially available oligomers prepared as methylated derivatives. As a component of this effort a set of software tools has been written for storing, organizing, and comparing spectral files, including the identification of isobaric mixtures. These tools provide a facile and objective evaluation of structural details including interresidue linkage, monomer identification, anomeric configuration, and branching. The tools are components of a web-based data sharing interface for sample tracking, spectral searching, and structural confirmation. Applications have been detailed with unknown samples and previously characterized glycoconjugates.The preceding report in this series presented data indicating that spectra from various stages of ion trap disassembly (MS n ) could supply structural details for the characterization of linkage, monomer ID, and branching. 1 With complete analysis as a practical consideration, there is a fundamental need for high-throughput tools to complement proteome studies. In that regard, little could be more important than searchable spectral library files for structural confirmation. The general reproducibility of ion trap (IT) spectra, and energy independence from the modes of ionization and collisional activation (CA), make compiling an MS n library an important research consideration.A number of oligosaccharide libraries have been reported each having specific characteristics applicable to the needs of independent research groups. GlycosidIQ 2 was developed for computerized interpretation of oligosaccharide spectra based on matching experimental data with theoretical fragments generated from GlycoSuiteDB, 3,4 a database of glycan structures reported in the literature. Similarly, the GlycoFragment/GlycoSearchMS package compares each peak of an observed mass spectrum with the calculated fragments of all structures contained in the SweetDB database to support the interpretation of mass spectra of complex carbohydrates. 5 Both GlycosidIQ and GlycoFragment/GlycoSearchMS utilize tandem MS (MS 2 ) of native samples and theoretically generated spectra as reference. Tseng et al. applied a "catalog library" approach for elucidating the structures of minor components in a mixture of oligosaccharides. 6 The catalog data consist of the characteristic fragmentation patterns belonging to a set of specific substructures compiled from a library of known compounds present in the same sample that have also been characterized by other techniques. Collisioninduced disassociation (CID) was used to determine the presence of the cataloged motifs. Recently, a standard trisaccharide MS 2 library has been reported that uses underivatized synthetic samples. 7 * Corresponding author. Tel: (603) In this report, we summarize initial progress and introduce simple ...

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.…”

A glycomics platform for the analysis of permethylated oligosaccharide alditols

“…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.…”

Discrimination of 16 structural isomers of fucosyl galactoside based on energy-resolved mass spectrometry

“…In most cases, there are differences in signal intensities, whereas in others, different fragment ions can be found. Accumulation of the spectral data in a database facilitates identification of oligosaccharides based on spectral matching [11][12][13][14][15][16][17]. An important problem-the elucidation of totally unknown structures that are not present in any databasewill still remain after the introduction of this efficient methodology.…”