College of Chemistry and Materials Science, Inner Mongolia Key Laboratory of Carbon Nanomaterials, Nano Innovation Institute (NII), Inner Mongolia Minzu University, Tongliao, 028000, PR China.
College of Chemistry and Materials Science, Inner Mongolia Key Laboratory of Carbon Nanomaterials, Nano Innovation Institute (NII), Inner Mongolia Minzu University, Tongliao, 028000, PR China.
Anal Chim Acta. 2023 Dec 1;1283:341974. doi: 10.1016/j.aca.2023.341974. Epub 2023 Oct 27.
Protein phosphorylation has been implicated in life processes including molecular interaction, protein structure transformation, and malignant disease. An in-depth study of protein phosphorylation may provide vital information for the discovery of early biomarkers. Mass spectrometry (MS)-based techniques have become an important method for phosphopeptide identification. Nevertheless, direct detection remains challenging because of the low ionization efficiency of phosphopeptides and serious interference from non-phosphopeptides. There is a great need for an efficient enrichment strategy to analyze protein phosphorylation prior to MS analysis.
In this study, a novel nanocomposite was prepared by introducing titanium ions into two-dimensional magnetic graphite nitride. The nanocomposite was combined with immobilized metal ion affinity chromatography (IMAC) and anion-exchange chromatography mechanisms for phosphoproteome research. The nanocomposite had the advantages of a large specific surface (412.9 m g), positive electricity (175.44 mV), and excellent magnetic property (35.7 emu g). Moreover, it presented satisfactory selectivity (α-casein:β-casein:bovine serum albumin = 1:1:5000), a low detection limit (0.02 fmol), great recyclability (10 cycles), and high recovery (92.8%). The nanocomposite demonstrated great practicability for phosphopeptides from non-fat milk, human serum, and saliva. Further, the nanocomposite was applied to enrich phosphopeptides from a more complicated specimen, A549 cell lysate. A total of 890 phosphopeptides mapping to 564 phosphoproteins were successfully detected with nano LC-MS.
We successfully designed and developed an efficient analysis platform for phosphopeptides, which includes protein digestion, phosphopeptide enrichment, and MS detection. The MS-based enrichment platform was further used to analyze phosphopeptides from complicated bio-samples. This work paves the way for the design and preparation of graphite nitride-based IMAC materials for phosphoproteome analysis.
蛋白质磷酸化参与了包括分子相互作用、蛋白质结构转化和恶性疾病在内的生命过程。深入研究蛋白质磷酸化可能为早期生物标志物的发现提供重要信息。基于质谱（MS）的技术已成为鉴定磷酸肽的重要方法。然而，由于磷酸肽的低离子化效率和非磷酸肽的严重干扰，直接检测仍然具有挑战性。因此，在 MS 分析之前，需要一种有效的富集策略来分析蛋白质磷酸化。
本研究通过将钛离子引入二维磁性石墨氮化碳制备了一种新型纳米复合材料。该纳米复合材料结合了固定化金属离子亲和色谱（IMAC）和阴离子交换色谱机制，用于磷酸蛋白质组学研究。该纳米复合材料具有大的比表面积（412.9 m g）、正电荷（175.44 mV）和优异的磁性（35.7 emu g）等优点。此外，它还具有令人满意的选择性（α-酪蛋白：β-酪蛋白：牛血清白蛋白=1:1:5000）、较低的检测限（0.02 fmol）、良好的可循环性（10 次）和较高的回收率（92.8%）。该纳米复合材料在非脂牛奶、人血清和唾液中的磷酸肽检测中表现出很好的实用性。此外，该纳米复合材料还应用于更复杂的 A549 细胞裂解物样本中磷酸肽的富集。通过纳升 LC-MS 成功检测到 890 个磷酸肽，对应 564 个磷酸蛋白。
我们成功设计并开发了一种用于磷酸肽分析的高效分析平台，包括蛋白质消化、磷酸肽富集和 MS 检测。该基于 MS 的富集平台进一步用于分析复杂生物样本中的磷酸肽。这项工作为基于石墨氮化碳的 IMAC 材料用于磷酸蛋白质组学分析的设计和制备铺平了道路。
