Preparation of Fe3O4@ZrO2 Core−Shell Microspheres as Affinity Probes for Selective Enrichment and Direct Determination of Phosphopeptides Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

• Published in: Journal of proteome research 2007-11, Vol.6 (11), p.4498-4510
• Main Authors: Li, Yan, Leng, Taohua, Lin, Huaqing, Deng, Chunhui, Xu, Xiuqing, Yao, Ning, Yang, Pengyuan, Zhang, Xiangmin
• Format: Article
• Language: English
• Subjects: Animals; Blood Proteins - chemistry; Caseins - chemistry; Cattle; Chickens; Chromatography, Liquid - methods; Ferric Compounds - chemistry; Horses; Hydrogen-Ion Concentration; Mass Spectrometry - methods; Microspheres; Phosphopeptides - chemistry; Proteomics - methods; Zirconium - chemistry
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr070167s

Fe3O4@ZrO2 microspheres with well-defined core−shell structure were prepared and applied for the highly selective enrichment of phosphopeptides from tryptic digest product of proteins. To successfully coat iron oxide microspheres with uniform zirconia shell, magnetic Fe3O4 microspheres were first synthesized via a solvothermal reaction, followed by being coated with a thin layer of carbon by polymerization and carbonization of glucose through hydrothermal reaction. Finally, with the use of the Fe3O4@C microspheres as templates, zirconium isopropoxide was prehydrolyzed and absorbed onto the microspheres and eventually converted into zirconia by calcinations. The as-prepared Fe3O4@ZrO2 core−shell microspheres were used as affinity probes to selectively concentrate phosphopeptides from tryptic digest of β-casein, casein, and five protein mixtures to exemplify their selective enrichment ability of phosphopeptides from complex protein samples. In only 0.5 min, phosphopeptides sufficient for characterization by MALDI-MS could be enriched by the Fe3O4@ZrO2 microspheres. The results demonstrate that Fe3O4@ZrO2 microspheres have the excellent selective enrichment capacity for phosphopeptides from complex samples. The performance of the Fe3O4@ZrO2 microspheres was further compared with commercial IMAC beads for the enrichment of peptides originating from tryptic digestion of β-casein and bovine serum albumin (BSA) with a molar ratio of 1:50, and the results proved a stronger selective ability of Fe3O4@ZrO2 microspheres over IMAC beads. Finally, the Fe3O4@ZrO2 microspheres were successfully utilized for enrichment of phosphopeptides from human blood serum without any other purification procedures. Keywords: Fe3O4@ZrO2 microspheres • Selective enrichment • MALDI-MS • Phosphopeptides