Voronina Liudmila, Fleischmann Frank, Šimunović Jelena, Ludwig Christina, Novokmet Mislav, Žigman Mihaela
Ludwig Maximilian University of Munich, Garching 85748, Germany.
Max Planck Institute of Quantum Optics, Garching 85748, Germany.
Anal Chem. 2024 Feb 7;96(7):2830-9. doi: 10.1021/acs.analchem.3c03589.
The health state of an individual is closely linked to the glycosylation patterns of his or her blood plasma proteins. However, obtaining this information requires cost- and time-efficient analytical methods. We put forward infrared spectroscopy, which allows label-free analysis of protein glycosylation but so far has only been applied to analysis of individual proteins. Although spectral information does not directly provide the molecular structure of the glycans, it is sensitive to changes therein and covers all types of glycosidic linkages. Combining single-step ion exchange chromatography with infrared spectroscopy, we developed a workflow that enables the separation and analysis of major protein classes in blood plasma. Our results demonstrate that infrared spectroscopy can identify different patterns and global levels of glycosylation of intact plasma proteins. To showcase the strengths and limitations of the proposed approach, we compare the glycoforms of human and bovine alpha-1-acid glycoproteins, which exhibit highly variable global levels of glycosylation. To independently evaluate our conclusions, the glycan moieties of human alpha-1-acid glycoprotein were further analyzed using an established glycomics workflow. Importantly, the chromatographic separation of blood plasma improves the detection of aberrant glycoforms of a given protein as compared to infrared spectroscopy of bulk plasma. The presented approach allows a time-efficient comparison of glycosylation patterns of multiple plasma proteins, opening new avenues for biomedical probing.
个体的健康状况与其血浆蛋白的糖基化模式密切相关。然而，获取此类信息需要具备成本效益和时间效益的分析方法。我们提出了红外光谱法，该方法可对蛋白质糖基化进行无标记分析，但迄今为止仅应用于单个蛋白质的分析。尽管光谱信息不能直接提供聚糖的分子结构，但它对其中的变化敏感，且涵盖了所有类型的糖苷键。我们将单步离子交换色谱法与红外光谱法相结合，开发了一种工作流程，能够对血浆中的主要蛋白质类别进行分离和分析。我们的结果表明，红外光谱法可以识别完整血浆蛋白的不同糖基化模式和整体水平。为了展示所提出方法的优势和局限性，我们比较了人和牛α-1-酸性糖蛋白的糖型，它们的整体糖基化水平差异很大。为了独立评估我们的结论，我们使用既定的糖组学工作流程对人α-1-酸性糖蛋白的聚糖部分进行了进一步分析。重要的是，与整体血浆的红外光谱相比，血浆的色谱分离提高了对给定蛋白质异常糖型的检测能力。所提出的方法能够高效地比较多种血浆蛋白的糖基化模式，为生物医学探索开辟了新途径。
