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Phosphopeptide enrichment for phosphoproteomic analysis - A tutorial and review of novel materials

Lookup NU author(s): Professor Phillip WrightORCiD


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© 2020 Elsevier B.V. Significant technical advancements in phosphopeptide enrichment have enabled the identification of thousands of p-peptides (mono and multiply phosphorylated) in a single experiment. However, it is still not possible to enrich all p-peptide species in a single step. A range of new techniques and materials has been developed, with the potential to provide a step-change in phosphopeptide enrichment. The first half of this review contains a tutorial for new potential phosphoproteomic researchers; discussing the key steps of a typical phosphoproteomic experiment used to investigate canonical phosphorylation sites (serine, threonine and tyrosine). The latter half then show-cases the latest developments in p-peptide enrichment including: i) Strategies to mitigate non-specific binding in immobilized metal ion affinity chromatography and metal oxide affinity chromatography protocols; ii) Techniques to separate multiply phosphorylated peptides from monophosphorylated peptides (including canonical from non-canonical phosphorylated peptides), or to simultaneously co-enrich other post-translational modifications; iii) New hybrid materials and methods directed towards enhanced selectivity and efficiency of metal-based enrichment; iv) Novel materials that hold promise for enhanced phosphotyrosine enrichment. A combination of well-understood techniques and materials is much more effective than any technique in isolation; but the field of phosphoproteomics currently requires benchmarking of novel materials against current methodologies to fully evaluate their utility in peptide based proteoform analysis.

Publication metadata

Author(s): Qiu W, Evans CA, Landels A, Pham TK, Wright PC

Publication type: Review

Publication status: Published

Journal: Analytica Chimica Acta

Year: 2020

Volume: 1129

Pages: 158-180

Print publication date: 08/09/2020

Online publication date: 28/04/2020

Acceptance date: 24/04/2020

ISSN (print): 0003-2670

ISSN (electronic): 1873-4324

Publisher: Elsevier B.V.


DOI: 10.1016/j.aca.2020.04.053