Rapid enrichment of phosphopeptides from tryptic digests of proteins using iron oxide nanocomposites of magnetic particles coated with zirconia as the concentrating probes

Chun Yuen Lo, Wei Yu Chen, Cheng Tai Chen, Yu-Chie Chen*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

132 Scopus citations

Abstract

Iron oxide nanocomposites of magnetic particles coated with zirconia were used as affinity probes to selectively concentrate phosphopeptides from tryptic digests of α- and β-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 β-casein using this approach is ∼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.

Original languageEnglish
Pages (from-to)887-893
Number of pages7
JournalJournal of Proteome Research
Volume6
Issue number2
DOIs
StatePublished - 1 Feb 2007

Keywords

  • Concentrating probes
  • Iron oxide
  • Magnetic particles
  • MALDI MS
  • Phospho-peptides
  • Zirconia

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