Profiling Analysis of Low Molecular Weight Heparins by Multiple Heart-Cutting Two Dimensional Chromatography with Quadruple Time-of-Flight Mass Spectrometry

Ouyang, Yilan; Zeng, Yangyang; Rong, Yinxiu; Song, Yue; Shi, Linjun; Chen, Bo; Yang, Xiaoguang; Xu, Nuo; Linhardt, Robert J.; Zhang, Zhenqing

doi:10.1021/acs.analchem.5b02218

Cited by 41 publications

(23 citation statements)

References 31 publications

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“…Ion-pair reversed phase chromatography (IPRP) is another powerful method for Hp/HS fractionation with the addition of ion-pairing agents, such as pentylamine 20 or tributylamine 21 . Compared with SAX, IPRP is more easily coupled with other separation methods and MS. Hyphenation of IPRP with SEC and time of flight mass spectrometry have greatly improved separation and provided more complete and important structural information of low molecular weight heparin (LMWH) 22 . But the ion-pair agents will easily induce in-source contamination and cause signal suppression when coupled with MS 13 .…”

Section: Introductionmentioning

confidence: 99%

Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis

Líu

Joshi

Chopra

et al. 2019

Preprint

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21Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which 22 are involved in diverse biological processes. The structural complexity brings 23 difficulties in separation, making the study of structure-function relationships 24challenging. Here we present a separation method for Hp/HS oligosaccharide 25 fractionation with cross-compatible solvent and conditions, combining size exclusion 26 chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and 27 hydrophilic interaction chromatography (HILIC) as three orthogonal separation 28 methods that do not require desalting or extensive sample handling. With this 29 method, the final eluent is suitable for structure-function relationship studies, 30including tandem mass spectrometry and microarray printing. Our data indicate that 31 high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, 32the fractions co-eluted in IPRP could be further separated by HILIC, with both 33 separation dimensions capable of resolving some isomeric oligosaccharides. We 34 demonstrate this method using both unpurified reaction products from isomeric 35 synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, 36 identifying isomers resolved by this multi-dimensional separation method. We 37 demonstrate both structural analysis by MS, as well as functional analysis by 38 microarray printing and screening using a prototypical Hp/HS binding protein: basic-39 fibroblast growth factor (FGF2). Collectively, this method provides a strategy for 40 efficient Hp/HS structure-function characterization . 41 42 43 44 45 46 47 48 Heparin and heparan sulfate (Hp/HS) are glycosaminoglycans (GAGs), highly 49 anionic unbranched polysaccharides found on the surface of essentially all 50 mammalian cells. Hp/HS consists of a repeating disaccharide structure either -D-51 glucuronic or -L-iduronic acid (GlcA and IdoA, respectively) and -D-N-acetyl-D-52 glucosamine (GlcNAc), all connected by 1→4 linkages 1 . As a part of biosynthesis, 53 monosaccharides can be differentially N-and O-sulfated after polymerization. The 54GlcA can be epimerized to IdoA and can be 2-O-sulfated, while the GlcNAc can be 55 deacetylated (usually followed by N-sulfation) and/or O-sulfated at the 6-and/or 3-56 position. Since these modifications are incomplete and untemplated, there is 57 enormous structural heterogeneity in Hp/HS chains, including many isomeric 58 structures with widely varying and dynamic compositions 2 . Different cell types will 59 often display different HS structures, and these structures can change as part of the 60 cell's physiological response 3 . This structural diversity mediates a wide range of 61 protein-GAG interactions of varying specificity and affinity. Hp/HS has been involved 62 in a wide and growing array of physiological and pathophysiological processes, 63usually mediated through interactions between proteins and a subset of Hp/HS 64 structures 4-6 . While the importance of oligosaccharide structure has been establishe...

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Section: Introductionmentioning

confidence: 99%

Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis

Líu

Joshi

Chopra

et al. 2019

Preprint

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“…Disaccharide analysis is still performed routinely to statistically determine the components and disaccharide backbone motifs of longer chains, but it results in a loss of structural information such as linkage, order, and sulfation patterns (147,148). coworkers to determine the major structures present (125,153,154). Over 80 compositions have been detected with these methods.…”

Section: Negative Electron Transfer Dissociation (Netd) Is Another Usmentioning

confidence: 99%

Developments in Mass Spectrometry for Glycosaminoglycan Analysis: A Review

Pepi

Sanderson

Stickney

et al. 2021

Molecular & Cellular Proteomics

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This review covers recent developments in glycosaminoglycan (GAG) analysis via mass spectrometry (MS). GAGs participate in a variety of biological functions, including cellular communication, wound healing, and anticoagulation, and are important targets for structural characterization. GAGs exhibit a diverse range of structural features due to the variety of O- and N-sulfation modifications and uronic acid C-5 epimerization that can occur, making their analysis a challenging target. Mass spectrometry approaches to the structure assignment of GAGs have been widely investigated, and new methodologies remain the subject of development. Advances in sample preparation, tandem MS techniques (MS/MS), on-line separations and automated analysis software have advanced the field of GAG analysis. These recent developments have led to remarkable improvements in the precision and time efficiency for the structural characterization of GAGs.

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“…In addition, an mRPLC-HILIC setup was able to resolve polar component that was poorly retained in the first dimension. Through mLC-LC consisting of SEC and ion-pairing RPLC, Ouyang et al 38 were able to identify and assign more than 80 and 120 oligosaccharides, respectively, from the low molecular weight heparins (LMWHs) nadroparin and enoxaparin.…”

Section: Applicability Of 2d-lcmentioning

confidence: 99%