Glycomic Analysis Using Glycoprotein Immobilization for Glycan Extraction

Yang, Shuang; Li, Yan; Shah, Punit; Zhang, Hui

doi:10.1021/ac400761e

Cited by 65 publications

(139 citation statements)

References 33 publications

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“…Many studies on N-glycans have shown that unmodified sialic acids are fragile and easily lost during sample preparation and ionization in MALDI or even ESI 20–24 . Chemical modification of sialic acids, such as amidation 22,25 , methyl esterification 26,27 and perbenzolylation 28 , is commonly used for sialic acid stabilization.…”

Section: Introductionmentioning

confidence: 99%

“…However, it is difficult to stabilize sialic acid residues using an in-solution chemical reaction because of the similarities between the chromatographic properties of these chemicals and those of the modified glycans. It is thus difficult to remove these excess chemicals from the modified glycans 20 . Permethylation of the released glycans can protect sialic acids on both N- and O-glycans 29,30 .…”

Section: Introductionmentioning

confidence: 99%

“…Our group has recently developed the GIG approach for the sequential release of N- and O-linked glycans 20 . GIG allows for the molecular stabilization of glycans on a solid support via chemoenzy-matic treatments.…”

Section: Introductionmentioning

confidence: 99%

“…As GIG depends on the conjugation of glycoproteins to a solid support, a high degree of conjugation efficiency is a prerequisite for quantitative analysis of glycans from samples. In general, the immobilization efficiency can be substantially improved (up to 95%) by adjusting the pH of the buffers 20,31 .…”

Section: Introductionmentioning

confidence: 99%

“…GIG has been used for the extraction of N-glycans for isobaric labeling using iARTs 51 or QUANTITY 37 . The fact that GIG takes advantage of a solid phase for sample preparation substantially reduces the loss of sample and eliminates the requirement for multiple chromatography for sample cleanup after each step 20,31 . The solid-phase resin can easily be packed into an AutoTip (pipette tip that holds packed beads for use in an automated liquid handler) for automated sample preparation (data not shown).…”

Section: Introductionmentioning

confidence: 99%

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Simultaneous quantification of N- and O-glycans using a solid-phase method

Yang

Sokoll

et al. 2017

Nat Protoc

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Glycosylation has a pivotal role in a diverse range of biological activities, modulating the structure and function of proteins. Glycogens coupled to the nitrogen atom (N-linked) of asparagine side chains or to the oxygen atom (O-linked) of serine and threonine side chains represent the two major protein glycosylation forms. N-glycans can be released by glycosidases, whereas O-glycans are often cleaved by chemical reaction. However, it is challenging to combine these enzymatic and chemical reactions in order to analyze both N- and O-glycans. We recently developed a glycoprotei n immobilization for glycan extraction (GIG) method that allows for the simultaneous analysis of N- and O-glycans on a solid support. GIG enables quantitative analysis of N-glycans and O-glycans from a single specimen and can be applied to a high-throughput automated platform. Here we provide a step-by-step GIG protocol that includes procedures for (i) protein immobilization on an aldehyde-active solid support by reductive amination; (ii) stabilization of fragile sialic acids by carbodiimide coupling; (iii) release of N-glycans by PNGase F digestion; (iv) release of O-glycans by β-elimination using ammonia in the presence of 1-phenyl-3-methyl-5-pyrazolone (PMP) to prevent alditol peeling from O-glycans; (v) mass spectrometry (MS) analysis; and (vi) data analysis for identification of glycans using in-house developed software (GIG Tool; free to download via http://www.biomarkercenter.org/gigtool). The GIG tool extracts precursor masses, oxonium ions and glycan fragments from tandem (liquid chromatography (LC)–MS/MS) mass spectra for glycan identification, and reporter ions from quaternary amine containing isobaric tag for glycan (QUANTITY) isobaric tags are used for quantification of the relative abundance of N-glycans. The GIG protocol takes ~3 d.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Simultaneous quantification of N- and O-glycans using a solid-phase method

Yang

Sokoll

et al. 2017

Nat Protoc

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Glycomic Analysis of Glycans Released from Glycoproteins Using Chemical Immobilization and Mass Spectrometry

Yang

Zhang

2014

CP Chemical Biology

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Protein glycosylation is one of most common protein modifications and is involved in many biological activities. N‐linked and O‐linked glycosylation not only represent abundant glycan modifications, but also are structurally diverse. Mass spectrometry has emerged as a major method for glycomic analysis. However, glycan extraction from proteins and glycan modification are two critical steps in glycomic analysis of glycans using mass spectrometry. In this protocol, we describe a novel and high‐throughput method for isolation and modification of glycans from glycoproteins using a chemoenzymatic approach on solid‐phase. Proteins are first immobilized to a solid support and unconjugated molecules are washed away; glycans, while still linked to glycoproteins on the solid support, can be treated enzymatically or chemically on solid phase for glycan derivatization. Glycans are then released from the solid support for analysis by mass spectrometry. The procedures outlined are robust and useful for high‐throughput glycomic analysis from complex biological or clinical samples. Curr. Protoc. Chem. Biol. 6:191‐208 © 2014 by John Wiley & Sons, Inc.

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Glycoprotein Biomarkers

Thomas

Yang

2017

Targeted Biomarker Quantitation by LC–MS

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Glycomic Analysis Using Glycoprotein Immobilization for Glycan Extraction

Cited by 65 publications

References 33 publications

Simultaneous quantification of N- and O-glycans using a solid-phase method

Simultaneous quantification of N- and O-glycans using a solid-phase method

Glycomic Analysis of Glycans Released from Glycoproteins Using Chemical Immobilization and Mass Spectrometry

Glycoprotein Biomarkers

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