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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