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Label-Free Method Development for Hydroxyproline PTM Mapping in Human Plasma Proteome

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Abstract

Post-translational modifications (PTMs) impart structural heterogeneities that can alter plasma proteins' functions in various pathophysiological processes. However, the identification and mapping of PTMs in untargeted plasma proteomics is still a challenge due to the presence of diverse components in blood. Here, we report a label-free method for identifying and mapping hydroxylated proteins using tandem mass spectrometry (MS/MS) in the human plasma sample. Our untargeted proteomics approach led us to identify 676 de novo sequenced peptides in human plasma that correspond to 201 proteins, out of which 11 plasma proteins were found to be hydroxylated. Among these hydroxylated proteins, Immunoglobulin A1 (IgA1) heavy chain was found to be modified at residue 285 (Pro285 to Hyp285), which was further validated by MS/MS study. Molecular dynamics (MD) simulation analysis demonstrated that this proline hydroxylation in IgA1 caused both local and global structural changes. Overall, this study provides a comprehensive understanding of the protein profile containing Hyp PTMs in human plasma and shows the future perspective of identifying and discriminating Hyp PTM in the normal and the diseased proteomes.

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Acknowledgement

This work has been carried out with financial assistance from the Science and Engineering Research Board, Government of India (GOI), File No. EMR/2016/002825. The authors acknowledge the Central Research Facility (CRF) of the Indian Institute of Technology Kharagpur (IIT Kgp) for establishing the MALDI facility. The authors acknowledge Rituparna Saha for correcting the manuscript. Debabrata, Gourab, and Shakilur thank IIT Kgp for individual fellowships.

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  1. Shakilur Rahman and Gourab Bhattacharje are these two authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India

    Debabrata Dutta, Shakilur Rahman, Gourab Bhattacharje & Amit Kumar Das

  2. School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India

    Swarnendu Bag & Jyotirmoy Chatterjee

  3. Central Research Facility, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India

    Bidhan Chandra Sing

  4. Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India

    Amit Basak

  5. School of Bioscience, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India

    Amit Basak & Amit Kumar Das

  6. Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India

    Debabrata Dutta

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  1. Debabrata Dutta
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Dutta, D., Rahman, S., Bhattacharje, G. et al. Label-Free Method Development for Hydroxyproline PTM Mapping in Human Plasma Proteome. Protein J 40, 741–755 (2021). https://doi.org/10.1007/s10930-021-09984-7

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