期刊
ANALYTICA CHIMICA ACTA
卷 636, 期 1, 页码 34-41出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.aca.2009.01.033
关键词
Monodisperse; Microspheres; Phosphopeptides enrichment; Phosphoproteome
资金
- National Natural Sciences Foundation of China [20675081, 20735004, 20605022, 90713017]
- China State Key Basic Research Program Grant [2005CB522701, 2007CB914102]
- China High Technology Research [2006AA02A309]
- Knowledge Innovation program of CAS [KJCX2.YW.HO9, KSCX2-YW-R-079]
- DICP
This study presented an approach to prepare monodisperse immobilized Ti4+ affinity chromatography (Ti4+-IMAC) microspheres for specific enrichment of phosphopeptides in phosphoproteome analysis. Monodisperse polystyrene seed microspheres with a diameter of ca. 4.8 mu m were first prepared by a dispersion polymerization method. Monodisperse microspheres with a diameter of ca. 13 mu m were prepared using the seed microspheres by a single-step swelling and polymerization method. Ti4+ ion was immobilized after chemical modification of the microspheres with phosphonate groups. The specificity of the Ti4+-IMAC microspheres to phosphopeptides was demonstrated by selective enrichment of phosphopeptides from Mixture of tryptic digests of alpha-casein and bovine serum albumin (BSA) at molar ratio of 1 to 500 by MALDI-TOF MS analysis. The sensitivity of detection for phosphopeptides determined by MALDI-TOF MS was as low as 5 fmol for standard tryptic digest of beta-casein. The Ti4+-IMAC microspheres were compared with commercial Fe3+-IMAC adsorbent and homemade Zr4+-IMAC microspheres for enrichment of phosphopeptides. The phosphopeptides and non-phosphopeptides identified by Fe3+-IMAC, Zr4+-IMAC and Ti4+-IMAC methods were 26,114,127 and 181,11,11 respectively for the same tryptic digest samples. The results indicated that the Ti4+-IMAC had the best performance for enrichment of phosphopepticles. (C) 2009 Elsevier B.V. All rights reserved.
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