Preparation of monodisperse immobilized Ti(4+) affinity chromatography microspheres for specific enrichment of phosphopeptides.
This study presented an approach to prepare monodisperse immobilized Ti(4+) affinity chromatography (Ti(4+)-IMAC) microspheres for specific enrichment of phosphopeptides in phosphoproteome analysis. Monodisperse polystyrene seed microspheres with a diameter of ca. 4.8mum were first prepared by a dispersion polymerization method. Monodisperse microspheres with a diameter of ca. 13mum were prepared using the seed microspheres by a single-step swelling and polymerization method. Ti(4+) ion was immobilized after chemical modification of the microspheres with phosphonate groups. The specificity of the Ti(4+)-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 5fmol for standard tryptic digest of beta-casein. The Ti(4+)-IMAC microspheres were compared with commercial Fe(3+)-IMAC adsorbent and homemade Zr(4+)-IMAC microspheres for enrichment of phosphopeptides. The phosphopeptides and non-phosphopeptides identified by Fe(3+)-IMAC, Zr(4+)-IMAC and Ti(4+)-IMAC methods were 26, 114, 127 and 181, 11, 11 respectively for the same tryptic digest samples. The results indicated that the Ti(4+)-IMAC had the best performance for enrichment of phosphopeptides.
Yu
Zhiyuan
Z
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
Han
Guanghui
G
Sun
Shutao
S
Jiang
Xinning
X
Chen
Rui
R
Wang
Fangjun
F
Wu
Ren'an
R
Ye
Mingliang
M
Zou
Hanfa
H
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D008863:Microspheres; D008969:Molecular Sequence Data; D010316:Particle Size; D010748:Phosphopeptides / Q000032:analysis* / Q000302:isolation & purification; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014025:Titanium / Q000737:chemistry*
D002364:Caseins; D010748:Phosphopeptides; D012710:Serum Albumin, Bovine; D014025:Titanium