標題: Rapid enrichment of phosphopeptides from tryptic digests of proteins using iron oxide nanocomposites of magnetic particles coated with zirconia as the concentrating probes
作者: Lo, Chun-Yuen
Chen, Wei-Yu
Chen, Cheng-Tai
Chen, Yu-Chie
應用化學系
應用化學系分子科學碩博班
Department of Applied Chemistry
Institute of Molecular science
關鍵字: zirconia;iron oxide;magnetic particles;phosphopeptides;MALDI MS;concentrating probes
公開日期: 2007
摘要: Iron oxide nanocomposites of magnetic particles coated with zirconia were used as affinity probes to selectively concentrate phosphopeptides from tryptic digests of alpha- and beta-caseins, milk, and egg white to exemplify the enrichment of phosphopeptides from complex samples. Phosphopeptides, in quantities sufficient for characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), were enriched by the affinity probes within only 30 s. The affinity probe-target species conjugates were separated from the sample solution simply by applying an external magnetic field. The detection limit for tryptic digest of beta-casein using this approach is similar to 45 fmol. Furthermore, we combined this enrichment method with a rapid enzymatic digestion method, that is, microwave-assisted enzymatic digestion using magnetic particles as the microwave absorbers, to speed up the tryptic digest reactions. Thus, we alternatively enriched phosphoproteins on the zirconia-coated particles followed by mixing with trypsin and heated the mixture in a microwave oven for 1 min. The particles remaining in the mixture were used as affinity probes to selectively enrich phosphopeptides from the tryptic digestion product by pipeting, followed by characterization using MALDI MS. Using the bifunctional zirconia-coated magnetic particles as both the affinity probes and the microwave absorbers could greatly reduce the time for the purification and characterization of phosphopeptides from complex samples.
URI: http://hdl.handle.net/11536/11153
http://dx.doi.org/10.1021/pr060333g
ISSN: 1535-3893
DOI: 10.1021/pr060333g
期刊: JOURNAL OF PROTEOME RESEARCH
Volume: 6
Issue: 2
起始頁: 887
結束頁: 893
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