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Enhanced N-glycosylation site exploitation of sialoglycopeptides by peptide IPG-IEF assisted TiO2 chromatography

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Abstract

Playing an important role in a broad range of biological and pathological processes, sialylation has been drawing wide interest. The efficient sialoglycopeptides enrichment methods are therefore attracting considerable attention. In this paper, we first compared two conventional enrichment methods, lectin and TiO2, and analyzed their characteristics. Furthermore, considering the highly negatively charged nature of sialic acids, we developed a new strategy, peptide immobilized pH gradient isoelectric focusing (IPG-IEF) assisted TiO2 chromatography (PIAT), for the highly efficient enrichment of sialoglycopeptides. In this method, peptides were first separated into 24 fractions using peptide IPG-IEF. Sialoglycopeptides were relatively concentrated in low-pH fractions of the immobilized pH strips and were captured using TiO2 chromatography. As a result, 614 N-glycosylation sites were identified in 582 sialoglycopeptides within 322 sialoglycoproteins from rat liver using PIAT. To our knowledge, this work represents one of the most comprehensive sialoglycoproteomic analyses in general and exhibits the largest database of sialoglycoproteome in rat liver currently. So the new strategy introduced here exhibits high efficiency and universality in the sialoglycopeptide enrichment, and is a powerful tool for sialoglycoproteome exploration.

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Abbreviations

PIAT:

Peptide IPG-IEF assistant TiO2 chromatography

SAs:

Sialic acids

MW:

Molecular weight

pI:

Isoelectric point

LC:

Liquid chromatography

MS/MS:

Tandem mass spectrometry

PNGase F:

N-glycosidase F

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Acknowledgments

This work was supported in part by National 973/S973/863 and NSF projects (S973-2011CB910600, NFS-31100590 and 20975024, S973-2010CB912700, 863-2012AA020200), and Shanghai Municipal Natural Science Foundation (11ZR1403000).

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Authors and Affiliations

  1. Institutes of Biomedical Sciences and Department of Chemistry, Fudan University, Shanghai, 200433, China

    Weiqian Cao, Jing Cao, Jiangming Huang, Lei Zhang, Jun Yao, Haoqi Xu & Pengyuan Yang

  2. Institutes of Biomedical Sciences and Department of Chemistry, Fudan University, Shanghai, 200032, China

    Pengyuan Yang

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  1. Weiqian Cao
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  2. Jing Cao
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  3. Jiangming Huang
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Correspondence to Pengyuan Yang.

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Weiqian Cao and Jing Cao contributed equally to this work.

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Cao, W., Cao, J., Huang, J. et al. Enhanced N-glycosylation site exploitation of sialoglycopeptides by peptide IPG-IEF assisted TiO2 chromatography. Glycoconj J 29, 433–443 (2012). https://doi.org/10.1007/s10719-012-9404-3

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