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Comparison and optimization of strategies for a more profound profiling of the sialylated N-glycoproteomics in human plasma using metal oxide enrichment

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Abstract

Glycosylation is an important posttranslational modification of proteins and plays a crucial role in both cellular functions and secretory pathways. Sialic acids (SAs), a family of nine-carbon-containing acidic monosaccharides, often terminate the glycan structures of cell surface molecules and secreted glycoproteins and perform an important role in many biological processes. Hence, a more profound profiling of the sialylated glycoproteomics may improve our knowledge of this modification and its effects on protein functions. Here, we systematically investigated different strategies to enrich the SA proteins in human plasma using a newly developed technology that utilizes titanium dioxide for sialylated N-glycoproteomics profiling by mass spectrometry. Our results showed that using a combination of a filter-aided sample preparation method, TiO2 chromatography, multiple enzyme digestion, and two-dimensional reversed-phase peptide fractionation led to a more profound profiling of the SA proteome. In total, 982 glycosylation sites in 413 proteins were identified, among which 37.8 % were newly identified, to establish the largest database of sialic acid containing proteins from human plasma.

Numbers of identified SA glycosites with different strategies

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Abbreviations

SA:

Sialic acid

FASP:

Filter-aided sample preparation

TiO2 :

Titanium dioxide

EGFR:

Epidermal growth factor receptor

RP:

Reversed-phase

LC-MS:

Liquid chromatography–mass spectrometry

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Acknowledgment

We are grateful for financial support from the National Key Program for Basic Research of China (2011CB910603, 2013CB911204); National High Technology Research and Development Program of China (2012AA020203, 2012AA020202); International Scientific Cooperation Project of China (2011DFB30370); and the National Natural Science Foundation of China (21275005,31100591,21235001), Beijing Nova Program (Z121107002512014).

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Authors and Affiliations

  1. College of Life Science and Bio-engineering, Beijing University of Technology, Beijing, 100022, China

    Xinyuan Zhao, Jifeng Wang & Xiaohong Qian

  2. State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, 102206, China

    Xinyuan Zhao, Cheng Ma, Huanhuan Han, Jing Jiang, Fang Tian, Wantao Ying & Xiaohong Qian

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  1. Xinyuan Zhao
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  2. Cheng Ma
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  3. Huanhuan Han
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  4. Jing Jiang
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  5. Fang Tian
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  6. Jifeng Wang
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  7. Wantao Ying
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Correspondence to Wantao Ying or Xiaohong Qian.

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Zhao, X., Ma, C., Han, H. et al. Comparison and optimization of strategies for a more profound profiling of the sialylated N-glycoproteomics in human plasma using metal oxide enrichment. Anal Bioanal Chem 405, 5519–5529 (2013). https://doi.org/10.1007/s00216-013-6971-5

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  • DOI: https://doi.org/10.1007/s00216-013-6971-5

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