Abstract

Magnetic materials in sample preparation for shotgun phosphoproteomics offer several advantages over conventional systems, as the enrichment can be achieved directly in solution, but they still suffer from some drawbacks, due to limited stability and selectivity, which is supposed to be affected by the hydrophilicity of the polymeric supports used for cation immobilization. The paper describes the development of an improved magnetic material with increased stability, thanks to a two-step covering of the magnetic core, for the enrichment of phosphopeptides in biological samples. Four materials were prepared featuring a polymeric shell with tunable hydrophilicity, obtained by “grafting from” polymerization of glycidyl methacrylate with 0–8.3% of polyethylene glycol methacrylate (PEGMA), the latter used to modulate the hydrophilicity of the material surface. Finally, the materials were functionalized with iminodiacetic acid for Ti4+ ion immobilization. The materials were analyzed for their composition by a combination of CHN elemental analysis and thermogravimetric analysis, also hyphenated to gas chromatography and mass spectrometric detection. Surface characteristics were evaluated by water contact angle measurements, scanning electron microscopy and energy dispersive X-ray spectrometry. These materials were applied to the enrichment of phosphopeptides from yeast protein digests. Peptides were identified by proteomics techniques using nano-high performance liquid chromatography coupled to mass spectrometry and bioinformatics. Qualitatively the peptides identified by the four systems were comparable, with 1606–1693 phosphopeptide identifications and a selectivity of 47–54% for all materials. The physico-chemical features of the identified peptides were also the same for the four materials. In particular, the grand average of hydropathy index values indicated that the enriched phosphopeptides were hydrophilic (ca. 90%), and only some co-enriched non-phosphorylated peptides were hydrophobic (21–28%), regardless of the material used for enrichment. Peptides had a pI ≤ 7, which indicated a well-known bias for acidic peptides binding, attributed to the interaction with the metal center itself. The results indicated that the enrichment of phosphopeptides and the co-enrichment of non-phosphorylated peptides is mainly driven by interactions with Ti4+ and does not depend on the amount of PEGMA chains in the polymer shell.

Highlights

  • Protein phosphorylation represents a major protein post translational modification and an active switch for several biological processes, a regulatory mechanism in physiological conditions whose dysfunction has been associated to several pathological conditions[1,2]

  • Modern phosphoproteomics comprises both untargeted and targeted approaches for phosphopeptide detection, the latter emerging as promising candidates to investigate site-specific phosphorylation[6], with both approaches relying on liquid chromatography coupled to tandem mass spectrometry (MS/MS)

  • Support hydrophilicity has been suggested to improve phosphopeptide enrichment selectivity for IMAC4,10–18, and for some metal oxide affinity chromatography (MOAC) materials[19,20,21,22], metal-organic framework (MOF) materials[23,24,25] and hybrid MOAC-IMAC materials as well[26]. This observation has prompted the development of hydrophilic materials for phosphopeptide enrichment; for instance, polydopamine was extensively used to prepare MOF23–25, IMAC supports[10,11,12,13,14,27,28,29,30], MOAC supports[31] or frameworks further derivatized with linkers, such as adenosine triphosphate[32]

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Summary

Introduction

Protein phosphorylation represents a major protein post translational modification and an active switch for several biological processes, a regulatory mechanism in physiological conditions whose dysfunction has been associated to several pathological conditions[1,2]. A multishell magnetic functional material was developed, starting from a promising one previously employed for phosphopeptide enrichment[33], and the preparation was modified to allow modulation of hydrophilic moieties displayed on the surface of the material. The four materials had www.nature.com/scientificreports similar amounts of Ti, which was responsible of the selective interaction with the phosphopeptides during enrichment.

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