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Evaluation of absorbent cotton for glycopeptide enrichment

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Abstract

Selecting proper and efficient glycopeptide enrichment approaches are essential for mass spectrometry-based glycoproteomics since glycopeptides are usually with microheterogeneity and low abundance in most biological samples. Herein, we introduced a cotton hydrophilic interaction liquid chromatography (HILIC) approach for large-scale glycopeptide enrichment with 80% acetonitrile/1% trifluoroacetic acid as the optimal sample loading buffer. The comparison of cotton HILIC with Venusil HILIC and mixed anion-exchange (MAX) approaches indicated that cotton HILIC was superior in overall glycopeptide enrichment, whereas Venusil HILIC preferred in complex glycan structures and MAX performed better with high mannose glycans. Exploration of capacity and recovery rate of cotton HILIC illustrated that 5mg cotton packed in a 200μL tip achieved a reasonable glycopeptide enrichment performance (~6% recovery) from ~0.5mg peptides. In conclusion, cotton HILIC can be used as an optional glycopeptide enrichment approach in glycosylation analysis with its specific merit.

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Data availability

The mass spectrometry data and results have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository [36] with the dataset identifier PXD035078 (username, reviewer_pxd035078@ebi.ac.uk; password, 4gDBebpX).

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Funding

This work was supported by the National Key R&D Program of China (2019YFA0905200), the Natural Sciences Foundation of Shaanxi Province (Grant No. 2021JQ-447), and the National Natural Science Foundation of China (Grant No. 91853123, 81773180, 81800655, and 21705127).

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Authors and Affiliations

  1. College of Life Science, Northwest University, Xi’an, Shaanxi Province, 710069, People’s Republic of China

    Miaomiao Xin, Shanshan You, Jingyu Wu, Yintai Xu, Cheng Li, Bojing Zhu, Jiechen Shen, Zexuan Chen, Liuyi Dang, Wei Dan & Shisheng Sun

  2. South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology, Faculty of Fisheries and Protection of Waters, University of South Bohemia in Ceske Budejovice, 38925, Vodnany, Czech Republic

    Miaomiao Xin

  3. Department of Medical Genetics, Xi’an Fourth Hospital, Xi’an, Shaanxi Province, 710004, People’s Republic of China

    Xinwen Zhang

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  1. Miaomiao Xin
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Correspondence to Shisheng Sun.

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Ethics approval

Mice-related experiments were conducted in compliance with ethical regulations and approved by the ethics committee of Northwest University, China. Human seminal plasma-related experiments were approved by the ethics committee of Xi’an Fourth Hospital and Northwest University.

Source of biological materials

Three mice, strain C57BL/6, male, aged 3 months, were obtained from the Central Animal Breeding House of Xi’an Jiaotong University, Xi’an, Shaanxi. Seminal plasma from six healthy men were collected in Xi’an Fourth Hospital. Semen was collected through masturbation in sterile containers after 3–7 days of sexual abstinence according to the criteria of the WHO laboratory manual. Then seminal plasma was obtained by centrifugation of semen.

Statement on animal welfare

Mice were anesthetized with intraperitoneal injection of chloral hydrate, and brain tissue was removed from each mouse and cut up in PBS buffer following internationally recognized guidelines.

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The authors declare no competing interests.

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Xin, M., You, S., Wu, J. et al. Evaluation of absorbent cotton for glycopeptide enrichment. Anal Bioanal Chem 414, 8245–8253 (2022). https://doi.org/10.1007/s00216-022-04353-4

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  • DOI: https://doi.org/10.1007/s00216-022-04353-4

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