Detection of endogenous peptides, especially those with modifications (such as phosphorylation) in biofluids, can serve as an indicator of intracellular pathophysiology. Although great progress has been made in phosphoproteomics in recent years, endogenous phosphopeptidomics has largely lagged behind. One main hurdle in endogenous phosphopeptidomics analysis is the coexistence of proteins and highly abundant nonmodified peptides in complex matrices. In this study, we developed an approach using zirconium(IV)-grafted mesoporous beads to enrich phosphopeptides, followed by analysis with a high resolution nanoRPLC-MS/MS system. The bifunctional material was first tested with digests of standard phosphoproteins and HeLa cell lysates, with excellent enrichment performance achieved. Given the size exclusion nature, the beads were directly applied for endogenous phosphopeptidomic analysis of serum samples from pancreatic ductal adenocarcinoma (PDAC) patients and controls. In total, 329 endogenous phosphopeptides (containing 113 high confidence sites) were identified across samples, by far the largest endogenous phosphopeptide data set cataloged to date. In addition, the method was readily applied for phosphoproteomics of the same set of samples, with 172 phosphopeptides identified and significant changes in dozens of phosphopeptides observed. Given the simplicity and robustness of the proposed method, we envision that it can be readily used for comprehensive phosphorylation studies of serum and other biofluid samples.

中文翻译：

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使用锆 (IV) 接枝介孔二氧化硅富集对血清进行深入的内源磷酸肽组学研究


内源肽的检测，尤其是生物液中经过修饰（例如磷酸化）的内源肽，可以作为细胞内病理生理学的指标。尽管近年来磷酸蛋白质组学取得了巨大进展，但内源磷酸肽组学却大大落后。内源磷酸肽组学分析的主要障碍之一是复杂基质中蛋白质和高丰度未修饰肽的共存。在本研究中，我们开发了一种使用锆 (IV) 接枝介孔珠来富集磷酸肽的方法，然后使用高分辨率 nanoRPLC-MS/MS 系统进行分析。该双功能材料首先使用标准磷蛋白消化物和 HeLa 细胞裂解物进行测试，取得了优异的富集性能。鉴于尺寸排阻性质，这些珠子直接用于胰腺导管腺癌 (PDAC) 患者和对照血清样本的内源性磷酸肽组学分析。总共，在样本中鉴定出了 329 个内源磷酸肽（包含 113 个高置信度位点），这是迄今为止编目的最大的内源磷酸肽数据集。此外，该方法还可轻松应用于同一组样品的磷酸化蛋白质组学，鉴定出 172 种磷酸肽，并观察到数十种磷酸肽的显着变化。鉴于所提出的方法的简单性和稳健性，我们设想它可以很容易地用于血清和其他生物流体样品的全面磷酸化研究。