期刊
JOURNAL OF PROTEOME RESEARCH
卷 7, 期 9, 页码 4164-4172出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr800120f
关键词
IMAC; titanium dioxide; phosphopeptide enrichment
Immobilized metal affinity chromatography (IMAC) based on Fe3+ or Ga3+ chelation is the most widely employed technique for the enrichment of phosphopeptides from biological samples prior to mass spectrometric analysis. An IMAC resin geared mainly toward phosphoprotein enrichment, Pro-Q Diamond, has been assessed for its utility in phosphopeptide isolation. Using both single phosphoprotein tryptic digests of beta-casein and ovalbumin and synthetic mixtures composed of tryptic digests of phosphorylated and nonphosphorylated protein standards, the selectivity and sensitivity of Pro-Q Diamond resin in an immobilized metal affinity-reversed phase microcolumn format were compared to an alternate titanium dioxide approach. The biphasic microcolumn method was found to be superior to metal oxide-based phosphopeptide capture in biological samples of increasing complexity. The lower limit of mass spectrometric detection for the immobilized metal affinity-reversed phase microcolumn approach was determined to be 10 pmol of beta-casein monophosphorylated peptide in 20 mu L of a solution of human serum protein digest (from 200 fig total serum protein after digestion and desalting).
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