Abstract
ABSTRACT Separation of phosphoproteins is essential for understanding their vital roles in biological processes and pathology. Transition metal-based receptors and antibodies, the routinely used materials for phosphoproteins enrichment, both suffer from low sensitivity, low recovery and coverage. In this work, a novel smart copolymer material was synthesized by modifying porous silica gel with a poly[(N-isopropylacrylamide-co-4-(3-acryloylthioureido) benzoic acid)0.35] (denoted as NIPAAm-co-ATBA0.35@SiO2). Driven by the hydrogen bonds complexation of ATBA monomers with phosphate groups, the copolymer-modified surface exhibited a remarkable adsorption toward native α-casein (a model phosphoprotein), accompanied with significant changes in surface viscoelasticity and roughness. Moreover, this adsorption was tunable and critically dependent on the polarity of carrier solvent. Benefiting from these features, selective enrichment of phosphoprotein was obtained using NIPAAm-co-ATBA0.35@SiO2 under a dispersive solid-phase extraction (dSPE) mode. This result displays a good potential of smart polymeric materials in phosphoprotein enrichment, which may facilitate top-down phosphoproteomics studies.
Highlights
Protein phosphorylation is a specific and reversible post-translational modification, which regulates numerous biological events, such as signal transduction, gene expression, and the cell cycle [1,2]
For discernment of the changes in surface morphology and roughness of the copolymer film before and after protein adsorption, the NIPAAm-co-ATBA-modified QC resonator was treated by a solution of protein (100 μg·mL−1 α-casein or bovine serum albumin (BSA) in CH3CN/H2O mixture with a volume ratio of 30:70) for 10 min at 20°C, which was followed by Atomic force microscopy (AFM) measurements in the scan mode under ambient condition [32]
A sharp decrease in fluorescence intensity of α-casein was observed upon the additions of various equivalents of ATBA. It was the fluorescence quenching caused by the binding of ATBA monomer and α-casein
Summary
Protein phosphorylation is a specific and reversible post-translational modification, which regulates numerous biological events, such as signal transduction, gene expression, and the cell cycle [1,2]. The bottom-up strategy comprises digestion of proteins, enrichment of phosphopeptides from peptide pool, mass spectrometry (MS) analysis and database searching [6]. Several materials have been developed to enrich phosphoproteins and to reduce the complexity of bio-samples [9,10,11,12] These materials mainly include species based on coordination interactions and multiple hydrogen bonding. We report a smart polymer-based material for phosphoprotein enrichment, which is constructed by immobilizing a poly[(N-isopropylacrylamide-co-4-(3acryloyl-thioureido) benzoic acid)0.35] (denoted as NIPAAm-co-ATBA0.35) onto the surface of porous silica gel. The porous silica gel with large surface area allows high grafting density of the smart copolymer Based on this design, the copolymer-modified silica gel (NIPAAm-co-ATBA0.35@SiO2) was successfully applied into the separation of α-casein and bovine serum albumin (BSA, a commonly used non-modified protein). The Ka value between ATBA and protein was obtained through a nonlinear fitting calculation according to the fluorescence intensity changes in the maximum emission peak (340 nm)
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