Preparation of Polypropylene Spin Tips Filled with Immobilized Titanium(IV) Ion Monolithic Adsorbent for Robust Phosphoproteome Analysis.
In this study, we developed a Ti(IV) monolithic spin tip for phosphoproteome analysis of a minute amount of biological sample for the first time. The surface of polypropylene pipet tip was activated by the photoinitiator benzophenone under UV light radiation followed by polymerization of ethylene glycol methacrylate phosphate and bis-acrylamide in the tip to form a porous monolith with reactive phosphate groups. The as-prepared tips grafted with monolithic adsorbent were then chelated with titanium(IV) ion for phosphopeptide enrichment. It was found that the tips enabled fast and efficient capture of phosphopeptides from microscale complex samples. The monolithic tip was demonstrated to have a detection limit as low as 5 fmol β-casein tryptic digest, along with an exceptionally high specificity to capture phosphopeptides from complex tryptic digest mixed with an unphosphorylated protein and a phosphorylated protein at a molar ratio up to 1000:1. When the tip was applied to enrich phosphopeptides from 5 μg of tryptic digest of complex HeLa cell proteins, 1185 high confidence of phosphorylated sites were successfully identified with the specificity as high as 92.5%. So far, this is the most sensitive phosphoproteomics analysis using a standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for proteome-wide phosphorylation analysis in mammalian cells.
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, 116023, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
Fangjie
F
Liu
Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology , Shanghai 200237, China.
Hao
H
Wan
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, 116023, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
Zhongshan
Z
Liu
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, 116023, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
Hongwei
H
Wang
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, 116023, China.|University of Chinese Academy of Sciences , Beijing 100049, China.
Jiawei
J
Mao
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, 116023, China.
Mingliang
M
Ye
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian, 116023, China.
Hanfa
H
Zou
D000178:Acrylamides / Q000737:chemistry; D000327:Adsorption; D001577:Benzophenones / Q000528:radiation effects; D002364:Caseins / Q000032:analysis; D002853:Chromatography, Liquid / Q000295:instrumentation* / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D057230:Limit of Detection; D008689:Methacrylates / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis; D010710:Phosphates / Q000737:chemistry; D010750:Phosphoproteins / Q000032:analysis*; D011126:Polypropylenes / Q000737:chemistry*; D016062:Porosity; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D053719:Tandem Mass Spectrometry / Q000295:instrumentation* / Q000379:methods; D014025:Titanium / Q000737:chemistry*; D014466:Ultraviolet Rays
D000178:Acrylamides; D001577:Benzophenones; D002364:Caseins; D008689:Methacrylates; D010446:Peptide Fragments; D010710:Phosphates; D010750:Phosphoproteins; D011126:Polypropylenes; D020543:Proteome; C047723:benzophenone; D014025:Titanium; C508958:ethylene glycol methacrylate phosphate