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A sequential separation strategy for facile isolation and comprehensive analysis of human urine N-glycoproteome

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Abstract

Urine is an attractive and non-invasive alternative source to tissue, blood or other biofluids for biomarker screening in clinical research. In normal human adult urine, 48% of the total urinary protein is in the sediment, 49% is soluble and the remaining 3% is contained in urinary extracellular vesicles (EVs). The soluble proteins and EV proteins in urine have attracted particular attention in recent years as cancer diagnostics. Furthermore, considering the important role of N-glycoproteins in practically all physiological processes, including regulating receptor-ligand binding, cell-cell interactions, inflammatory response and tumour progression, N-glycoproteome in human urine is an invaluable target for monitoring the physiological status and pathological changes of the kidney and urinary tract. Given the different origins of the soluble proteins and EV proteins in the urine, different N-glycoproteome patterns exist. Therefore, isolating the soluble N-glycoproteins and EV N-glycoproteins for separate analysis will provide a more specific and comprehensive view and provide a deeper understanding of human urinary N-glycoproteome. In this work, we developed a sequential separation method that isolates urinary soluble proteins and EV proteins via stepwise ultrafiltration based on their obvious size difference. A facile and reproducible protein isolation was achieved using this strategy. Subsequent N-glycoproteome enrichment and identification revealed distinct patterns in the two sub-proteomes of urine with more than 60% differential N-glycopeptides. A more comprehensive picture of the urinary N-glycoproteome with close to 1800 identified N-glycopeptides was obtained by this new analysis strategy, therefore making it advantageous for urinary biomarker screening.

A sequential separation method that isolates urinary soluble proteins and EV proteins via stepwise ultrafiltration was developed in this work. Subsequent N-glycopeptides enrichment and mass spectrometry analysis reveals distinct N-glycoproteome patterns in the two sub-proteomes of urine and a deep mapping of close to 1800 N-glycopeptides.

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Acknowledgments

This study was supported by the National Key Program for Basic Research of China (No. 2017YFC0906703, 2016YFA0501403, 2018YFF0212505, 2017YFA0505002), National Natural Science Foundation of China (No. 21675172), National Key Laboratory of Proteomics Grant (SKLP-K201706), BPRC-Tianjin Baodi Hospital Joint Center Grant (TMRC2014Z03), Innovation Project (16CXZ027) and Beijing Science and Technology Plan Project (No. Z161100002616036).

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Authors and Affiliations

  1. Research Center for Analytical Sciences, College of Sciences, Northeastern University, Shenyang, 110819, Liaoning, China

    Bianbian Huo, Mingli Chen, Junjie Chen, Yuanyuan Li & Jianhua Wang

  2. State Key Laboratory of Proteomics, National Center for Protein Sciences (Beijing), Beijing Proteome Research Center, Beijing Institute of Lifeomics, Beijing, 102206, China

    Bianbian Huo, Junjie Chen, Yuanyuan Li, Wanjun Zhang, Weijie Qin & Xiaohong Qian

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  1. Bianbian Huo
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Correspondence to Wanjun Zhang, Jianhua Wang or Weijie Qin.

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The authors state that all experiments were performed in compliance with the relevant laws and institutional guidelines. The institutional committee of the National Centre for Protein Science Beijing has approved the experiments with live subjects. The authors also state that informed consent was obtained for any experimentation with human subjects and National Centre for Protein Science Beijing is committed to the protection and safety of human subjects involved in research.

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Huo, B., Chen, M., Chen, J. et al. A sequential separation strategy for facile isolation and comprehensive analysis of human urine N-glycoproteome. Anal Bioanal Chem 410, 7305–7312 (2018). https://doi.org/10.1007/s00216-018-1338-6

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  • DOI: https://doi.org/10.1007/s00216-018-1338-6

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