Synthesis of Thermally Switchable Chromatographic Materials with Immobilized Ti4+ for Enrichment of Phosphopeptides by Reversible Addition Fragmentation Chain Transfer Polymerization

2017 3rd International Conference on Environmental Science and Material Application

生态环境科学;材料科学

Wang, Di^1 ; Cao, Zhihan^1 ; Pang, Xinzhu^1 ; Deng, Yulin^1 ; Li, Bo^2 ; Dai, Rongji^1

Beijing Key Labrotary for Separation and Analysis in Biomedicine and Pharmaceuticals, School of Life Science, Beijing Institute of Technology, Beijing

100081, China^1

Advanced Research Institute of Multidisciplinary Science, Beijing Institute of Technology, Beijing

100081, China^2

关键词: Enrichment of phosphopeptides;    IMAC (immobilized metal affinity chromatography);    Poly-n-isopropyl acrylamide;    Post-translational modifications;    Pyridoxal 5' phosphates;    Reversible addition-fragmentation chain transfer polymerization;    Reversible phosphorylation;    Temperature-responsive materials;   

Others  :  https://iopscience.iop.org/article/10.1088/1755-1315/108/2/022018/pdf
DOI  :  10.1088/1755-1315/108/2/022018

来源: IOP

【 摘 要 】

Reversible phosphorylation of proteins is one of the most crucial types of post-translational modifications (PTMs). And it shows significant work in diversified biological processes. However, the separation technology of phosphorylated peptides is still an analytical challenge in phosphoproteomics, because phosphopeptides are alway in low stoichiometry. Thus, enrichment of phosphopeptides before detection is indispensable. In this study, a novel temperature regulated separation protocol was developed. Silica@p (NIPAAm-co-IPPA)-Ti4+, a new Ti(IV)-IMAC (Immobilized Metal Affinity chromatography) materials was synthesized by reversible addition fragmentation chain transfer polymerization (RAFT). By the unique thermally responsive properties of poly(N-isopropylacrylamide) (PNIPAAm), the captured phosphorylated peptides could be released by changing temperature only without applying any other eluant which could damage the phosphopeptides. We employed isopropanol phosphonic acid (IPPA) as an IMAC ligand for the immobilization of Ti(IV) which could increase the specific adsorption of phosphopeptides. The enrichment and release properties were examined by treatment with pyridoxal 5'-phosphate (PLP) and casein phosphopeptides (CPP). Two phosphorylated compounds above have temperature-stimulated binding to Ti4+. Finally, silica@p (NIPAAm-co-IPPA)-Ti4+was successfully employed in pretreatment of phosphopeptides in a tryptic digest of a-casein and human serum albumin (HSA). The results indicated a great potential of this new temperature-responsive material in phosphoproteomics study.