梗概蛋白质翻译后修饰（PTMs）调控几乎所有细胞过程。异常的PTMs与多种人类疾病密切相关，这大大复杂了特定疾病与基因突变的关联，甚至使基因组驱动的个体化治疗无效。PTM蛋白质组学具有提供对疾病生物学更全面理解的潜力，并正在成为后基因组学时代精准医学的新突破之一。作为最突出的两种PTMs，磷酸化和糖基化在学术界吸引了广泛的研究兴趣，用于发现癌症发病机制、生物标志物和治疗靶点，以及在工业界开发靶向药物和疫苗方面。尽管利益可观，目前的磷酸化和糖基化分析仍面临很大挑战，因为磷酸化和糖基化的含量极低，而且糖基化具有极大的异质性和结构复杂性。迄今为止，我们的实验室专注于蛋白质磷酸化和糖基化，并为创新分离和分析工具的开发提供了独特的见解。本论文概述了这些化学工具，以及我们对蛋白质磷酸化和糖基化分析挑战的潜在解决方案的看法。 第一部分描述了我们在磷酸化富集和检测方面的努力。鉴于智能聚合物的良好响应性和可扩展性，我们设计了一种带有亲和配体的智能聚合物材料，通过可调捕获和释放实现了多重磷酸化肽的高效富集。此外，我们还介绍了将带有配体的智能聚合物与固态纳米通道结合以创建功能性纳米流体设备的方法。这些设备可以监测激酶反应，从而为激酶活性提供了一种低成本、无标记的测定方法。在接下来的部分中，我们讨论了糖基化物种的捕获策略和糖基分析方法。通过一种水解反应，发现含有席夫碱基的材料可以高效地捕获唾液酸化糖肽，展示了动态共价化学在富集中的威力。然后，基于噬菌体展示的定向进化策略被用来开发靶向脂多糖（LPS）的配体，在聚合物中作为吸附剂，可以高效清除循环中的LPS。基于纳米流体设备对简单糖基异构体的区分能力，我们通过衍生化策略进一步探索了利用蛋白质纳米孔识别多样化糖基的方法。这一探索的成功为推进基于纳米孔的单分子糖基分析和糖基测序提供了解决方案。虽然不是详尽无遗，但本论文可以为化学工具的开发，以促进对磷酸化和糖基化等PTMs的分析提供新的见解。

ConspectusProtein post-translational modifications (PTMs) modulate almost all cellular processes. Aberrant PTMs are closely bound to various human diseases, which greatly complicates the association of a specific disease with a genetic mutation and even renders genomics-driven personalized therapeutics ineffective. PTM proteomics has the potential to provide a more comprehensive understanding of disease biology and is emerging as one of the new breakthroughs of precision medicine in the postgenomics era. As two of the most prominent PTMs, phosphorylation and glycosylation have attracted broad research interest both in academia for the discovery of cancer pathogenesis, biomarkers, and therapeutic targets and in industry for the development of targeted drugs and vaccines. Despite the great rewards, current phosphorylation and glycosylation analyses are still faced with some considerable challenges due to the ultralow abundance of phosphorylation and glycosylation and the grand heterogeneity and structural complexity of glycosylation. To date, our laboratory has concentrated on protein phosphorylation and glycosylation and offered unique insights for the development of innovative separation and analytical tools. An overview of these chemical tools, together with our views on potential solutions to the challenges of protein phosphorylation and glycosylation analysis, is presented in this Account.The first part describes our efforts in phosphorylation enrichment and sensing. Given the excellent responsiveness and expandability of smart polymers, we designed a smart polymer material bearing affinity ligands that target the phosphate group and achieved efficient enrichment of multiply phosphorylated peptides through a tunable catch-and-release. Further, the combination of ligand-bearing smart polymers with solid-state nanochannels for the creation of functional nanofluidic devices is presented. The devices allow us to monitor the kinase reaction, thus providing a low-cost, label-free assay for kinase activity. In the next section, we discuss capture strategies for glycosylated species and glycan analysis methods. A Schiff base-containing material was revealed to achieve high-efficiency capture of sialylated glycopeptides through a hydrolysis reaction, demonstrating the power of dynamic covalent chemistry for enrichment. Then, a directed evolution strategy based on phage display was used to develop a lipopolysaccharide (LPS)-targeted ligand that, when incorporated into a polymer as an adsorbent, can efficiently clear the circulating LPS. Based on the distinction of simple glycan isomers with a nanofluidic device, we further explored the identification of diverse glycans with a protein nanopore through a derivatization strategy. The success of the exploration offers a solution to advance nanopore-based single-molecule glycan profiling and glycan sequencing. Although not comprehensive, this Account could provide new insight into the development of chemical tools to facilitate the analysis of phosphorylation and glycosylation as well as other PTMs.