Abstract
Multisite phosphorylation is an important and common mechanism for finely regulating protein functions and subsequent cellular responses. However, this study is largely restricted by the difficulty to capture low-abundance multiply phosphorylated peptides (MPPs) from complex biosamples owing to the limitation of enrichment materials and their interactions with phosphates. Here we show that smart polymer can serve as an ideal platform to resolve this challenge. Driven by specific but tunable hydrogen bonding interactions, the smart polymer displays differential complexation with MPPs, singly phosphorylated and non-modified peptides. Importantly, MPP binding can be modulated conveniently and precisely by solution conditions, resulting in highly controllable MPP adsorption on material surface. This facilitates excellent performance in MPP enrichment and separation from model proteins and real biosamples. High enrichment selectivity and coverage, extraordinary adsorption capacities and recovery towards MPPs, as well as high discovery rates of unique phosphorylation sites, suggest its great potential in phosphoproteomics studies.
Highlights
Multisite phosphorylation is an important and common mechanism for finely regulating protein functions and subsequent cellular responses
We designed an H-bondingbased phosphate receptor, 4-(3-acryloyl-thioureido)-benzoic acid (ATBA; Supplementary Figs. 1, 2), to copolymerize withNIPAAm to form a linear random copolymer through atom transfer radical polymerization[32] (ATRP; the detailed preparation method is described in Supplementary Methods)
In summary, above results reveal four attracting features of our material: specific and tunable complexation with multiply phosphorylated peptides (MPPs) driven by multiple H-bonding interaction, smart adsorption conversion windows modulated by solvent polarity, solution pH or temperature, high adsorption capacities and recovery, as well as extraordinary enrichment capacities towards diverse MPPs from real biosamples
Summary
Multisite phosphorylation is an important and common mechanism for finely regulating protein functions and subsequent cellular responses. This study is largely restricted by the difficulty to capture low-abundance multiply phosphorylated peptides (MPPs) from complex biosamples owing to the limitation of enrichment materials and their interactions with phosphates. MPP binding can be modulated conveniently and precisely by solution conditions, resulting in highly controllable MPP adsorption on material surface This facilitates excellent performance in MPP enrichment and separation from model proteins and real biosamples. By incorporating appropriate biomolecule-recognition units into PNIPAAm, the polymer conformation may be intelligently modulated using guest biomolecules, dramatic conformational transition of the polymer chains will in turn remarkably influence the binding or release of the guest biomolecules[29,30,31] Such copolymer may provide an efficient tool to modulate the dynamic adsorption/ desorption behaviours of MPPs on material surfaces, which suggests an ideal platform for MPP enrichment and separation
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