Simple separation of isomeric sialylated N‐glycopeptides by a zwitterionic type of hydrophilic interaction chromatography

Takegawa, Yasuhiro; Deguchi, Kisaburo; Ito, Hiroki; Keira, Takuro; Nakagawa, Hiroaki; Nishimura, Shin‐Ichiro

doi:10.1002/jssc.200600133

Cited by 137 publications

(111 citation statements)

References 33 publications

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“…Similar effects are evident in the separation of N-glycans on the sulfobetaine (ZIC-HILIC) column. 60 While Takegawa et al provided a fine explanation of the effect of electrostatic repulsion on retention of sialylated glycans, they had some difficulty accounting for the slight increase in retention of 2-aminopyridine (PA)-derivatized neutral glycans when the ammonium acetate concentration is increased from 5 to 20 mM. At 5 mM salt, one can assume that the sulfonate group in the coating is in a zwitterionic interaction with the embedded amine group.…”

Section: Discussionmentioning

confidence: 99%

Electrostatic Repulsion Hydrophilic Interaction Chromatography for Isocratic Separation of Charged Solutes and Selective Isolation of Phosphopeptides

Alpert¹

2007

Anal. Chem.

491

373

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If an ion-exchange column is eluted with a predominantly organic mobile phase, then solutes can be retained through hydrophilic interaction even if they have the same charge as the stationary phase. This combination is termed electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). With mixtures of solutes that differ greatly in charge, repulsion effects can be exploited to selectively antagonize the retention of the solutes that normally would be the best retained. This permits the isocratic resolution of mixtures that normally require gradients, including peptides, amino acids, and nucleotides. ERLIC affords convenient separations of highly charged peptides that cannot readily be resolved by other means. In addition, phosphopeptides can be isolated selectively from a tryptic digest.Solutes in a mixture can differ greatly in their properties. In reversed-phase chromatography (RPC), this involves differences in polarity. For ion-exchange chromatography, this involves differences in charge. An elution gradient of some sort is generally used to ensure that all solutes in a mixture elute in the same time frame. This paper introduces an alternative strategy: superimposing a second mode of chromatography that selectively reduces the retention of the solutes that are usually the most strongly retained. The combination used here is ion-exchange and hydrophilic interaction.The term hydrophilic interaction chromatography (HILIC) was coined in 1990 1 to describe normal-phase chromatography with mobile phases that, typically, are 10-40% aqueous. A sufficiently polar stationary-phase material is more polar than this mobile phase and will retain polar solutes. A model for the retention mechanism postulates partitioning between the dynamic mobile phase and a slow-moving layer of water with which the polar stationary phase is hydrated. The more polar a solute, the more it associates with this stagnant aqueous phase and the later it elutes, a normal-phase direction. HILIC was used for carbohydrate analysis as early as 1975. 2,3 The mechanism of separation was recognized as early as 1967, in the case of Sephadex eluted with a predominantly organic mobile phase. 4 HILIC is useful for analysis of polar solutes in general as RPC is for nonpolar solutes. Since 1990, HILIC has been applied to a wide variety of peptides, 5-10 complex carbohydrates, 11 and some proteins 12-15 and is increasingly being applied to small polar solutes such as pharmaceuticals, [16][17]

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Section: Discussionmentioning

confidence: 99%

Electrostatic Repulsion Hydrophilic Interaction Chromatography for Isocratic Separation of Charged Solutes and Selective Isolation of Phosphopeptides

Alpert¹

2007

Anal. Chem.

491

373

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“…A better approach to glycopeptide or protein enrichment may be the application of hydrophobic interaction chromatography (HILIC). HILIC has proven to be a convenient method for analysing highly polar molecules such as metabolites and it has been adapted to be efficient in the extraction of glycopeptides [33]. Many glycobiology studies are using HILIC and one recent study has applied the HILIC method to the glyco-profiling of a therapeutic monoclonal antibody and proteins with several N-linked and O-linked glycosylation sites.…”

Section: Glycopeptidomicsmentioning

confidence: 99%

The Emerging Field of Diagnostics in Glycosylation Disorders

Heywood¹,

Clayton²,

Mills³

et al. 2013

Pediat Therapeut

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Protein glycosylation is an important post-translational modification. It enhances the functional diversity of proteins, half-life and influences their biological activity. Defective glycosylation often leads to multisystem disease and adds itself to the expanding group of 'Congenital disorders or glycosylation' which are predominantly disorders of N-linked glycosylation. Another rapidly growing group of disorders are defects in O-linked glycosylation, including a subset of dystroglycanopathies. Current diagnostic strategies for glycosylation disorders are compounded by the multivariate clinical phenotype of many of the diseases. Biochemical tests such as the isoelectric focusing of transferrin and apolipoprotein CIII are used to assess a patient's glycoform profile before in depth enzyme and genetic analysis is initiated. Whilst the glycoform profiling has been instrumental in screening for many glycosylation disorders, there is a need for a more sensitive and informative test. This short review gives an overview of the recent methods used in glycobiology research that could be used to devise such a test, which alongside currently used diagnostic tests should further facilitate the delineation of CDG subtypes. It provides a view to a potential strategy using marker glycopeptides to develop a mass spectrometry based assay that could be implemented into clinical diagnostic laboratories.

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“…In particular, the puriˆca-tion/separation of isomeric glycans must be performed very carefully since only an MS n spectral library of pure standards (quality) can be used for MS n spectral matching, and also for the determination of the mixing ratio of isomers (30). (4) HPLC columns are highly capable of distinguishing between the structures of isomeric glycans (54)(55)(56)(57). Thus, the HPLC retention times of glycans also provide very useful information for the assignment of these molecules.…”

Section: Issues To Be Addressedmentioning

confidence: 99%

Untitled

Deguchi

2008

TIGG

Self Cite

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Key Words: oligosaccharides, mass spectrometry, spectral library,glycopeptides A. IntroductionDNA sequences are now routinely analyzed by using highthroughput polymerase chain reaction (PCR) and DNA sequencers. Trace analyses of proteins are also being performed by combining highly selective and sensitive mass spectrometry (MS) with a universal protein database (e.g., SWISS-PROT) (1). However, for oligosaccharides (glycans), MS-based analysis continues to be associated with many di‹culties despite the fact that the molecular weights of glycans (a few hundred to a few thousand Daltons) are considerably smaller than are those of proteins and DNA. The reason for such di‹culties stems from the structural diversity of these molecules and the existence of structural isomers, the latter of which are inherent features of glycans that arise from various glycoside linkages, branching, anomeric (a, b) conˆgurations, and monosaccharide isomers. Isomers pose various problems for MS analysis, a technique that is based on the measurement of ion mass (m/z: mass-to-charge ratio). A further di‹culty is related to the heterogeneity of the N-and O-glycans attached to proteins There are, for example, 16 diŠerent types of neutral N-glycan associated with the human IgG protein (2). Consequently, the amount of each glycan is considerably smaller than that of the parent protein. Therefore, analysis of glycans requires an appreciably more sensitive MS and a much more meticulous sample preparation in order to minimize glycan loss (3-5).Last year, MS-based analytical methods for glycans and glycoproteins (i.e., glycomics and glycoproteomics) were widely reviewed in this journal (6,7). Although some duplication of this material is unavoidable, I would like to review the problems associated with current MS techniques, and in particular focus on the assignment of glycans based on MS n spectral database and matching. I will also brie‰y describe the future prospects for these techniques. B. MS-based structural analysis of glycansGlycans containing many hydroxyl groups are nonvolatile

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Simple separation of isomeric sialylated N‐glycopeptides by a zwitterionic type of hydrophilic interaction chromatography

Cited by 137 publications

References 33 publications

Electrostatic Repulsion Hydrophilic Interaction Chromatography for Isocratic Separation of Charged Solutes and Selective Isolation of Phosphopeptides

Electrostatic Repulsion Hydrophilic Interaction Chromatography for Isocratic Separation of Charged Solutes and Selective Isolation of Phosphopeptides

The Emerging Field of Diagnostics in Glycosylation Disorders

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