Phosphorylation is a form of protein posttranslational modification (PTM) that regulates many biological processes. Whereas phosphoproteomics is a scientific discipline that identifies and quantifies the phosphorylated proteome using mass spectrometry (MS). This task is extremely challenging as ~30% of the human proteome is phosphorylated; and each phosphoprotein may exist as multiple phospho-isoforms that are present in low abundance and stoichiometry. Hence, phosphopeptide enrichment techniques are indispensable to (phospho)proteomics laboratories. These enrichment methods encompass widely-adopted techniques such as (i) affinity-based chromatography; (ii) ion exchange and mixed-mode chromatography (iii) enrichment with phospho-specific antibodies and protein domains, and (iv) functionalized polymers and other less common but emerging technologies such as hydroxyapatite chromatography and precipitation with inorganic ions. Here, we review these techniques, their history, continuous development and evaluation. Besides, we outline associating challenges of phosphoproteomics that are linked to experimental design, sample preparation, and proteolytic digestion. In addition, we also discuss about the future outlooks in phosphoproteomics, focusing on elucidating the noncanonical phosphoproteome and deciphering the “dark phosphoproteome”. © 2020 John Wiley & Sons Ltd.

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扩大磷酸化蛋白质组学的瓶颈：不断发展的磷酸化富集策略

磷酸化是蛋白质翻译后修饰 (PTM) 的一种形式，可调节许多生物过程。而磷酸化蛋白质组学是一门科学学科，它使用质谱法 (MS) 识别和量化磷酸化蛋白质组。这项任务极具挑战性，因为大约 30% 的人类蛋白质组被磷酸化；并且每种磷蛋白可以以低丰度和化学计量存在的多种磷酸亚型存在。因此，磷酸肽富集技术对于（磷酸）蛋白质组学实验室是必不可少的。这些富集方法包括广泛采用的技术，例如 (i) 基于亲和的色谱法；(ii) 离子交换和混合模式色谱 (iii) 富集磷酸化特异性抗体和蛋白质结构域，(iv) 功能化聚合物和其他不太常见但新兴的技术，如羟基磷灰石色谱和无机离子沉淀。在这里，我们回顾了这些技术、它们的历史、持续发展和评估。此外，我们概述了与实验设计、样品制备和蛋白水解消化相关的磷酸化蛋白质组学的相关挑战。此外，我们还讨论了磷酸化蛋白质组学的未来前景，重点是阐明非典型磷酸化蛋白质组和破译“暗磷酸化蛋白质组”。© 2020 约翰威利父子公司。我们概述了与实验设计、样品制备和蛋白水解消化相关的磷酸化蛋白质组学的相关挑战。此外，我们还讨论了磷酸化蛋白质组学的未来前景，重点是阐明非典型磷酸化蛋白质组和破译“暗磷酸化蛋白质组”。© 2020 约翰威利父子公司。我们概述了与实验设计、样品制备和蛋白水解消化相关的磷酸化蛋白质组学的相关挑战。此外，我们还讨论了磷酸化蛋白质组学的未来前景，重点是阐明非典型磷酸化蛋白质组和破译“暗磷酸化蛋白质组”。© 2020 约翰威利父子公司。