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.

翻译后修饰 (PTM) 赋予结构异质性，可以改变血浆蛋白在各种病理生理过程中的功能。然而，由于血液中存在多种成分，非靶向血浆蛋白质组学中 PTM 的识别和定位仍然是一个挑战。在这里，我们报告了一种使用串联质谱 (MS/MS) 在人血浆样品中识别和绘制羟基化蛋白质的无标记方法。我们的非靶向蛋白质组学方法使我们在人血浆中鉴定了 676 个从头测序的肽，这些肽对应于 201 种蛋白质，其中 11 种血浆蛋白质被发现被羟基化。在这些羟基化蛋白质中，发现免疫球蛋白 A1 (IgA1) 重链在残基 285（Pro ²⁸⁵到 Hyp ²⁸⁵)，这得到了 MS/MS 研究的进一步验证。分子动力学 (MD) 模拟分析表明 IgA1 中的脯氨酸羟基化导致局部和全局结构变化。总的来说，这项研究提供了对人血浆中含有 Hyp PTM 的蛋白质谱的全面了解，并展示了在正常和患病蛋白质组中识别和区分 Hyp PTM 的未来前景。