Enhancement of Ionization Efficiency and Selective Enrichment of Phosphorylated Peptides from Complex Protein Mixtures Using a Reversible Poly-Histidine Tag

Pegah R. Jalili, Deepti Sharma, Haydn L. Ball

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

To improve the detection of phosphorylated peptides/proteins, a combination of optimized MS-based strategies were used involving chemical derivatization with a polyhistidine-tag (His-tag) and affinity enrichment of the resulting His-tag peptides on a nanoscale Ni2+-IMAC column. The phosphoserine and phosphothreonine peptides were derivatized using a one-pot β-elimination/Michael addition reaction with a reversible His-tag possessing a thiol-containing Cys residue. The His-tag peptides were enriched selectively by Ni2+-IMAC and released using either imidazole or cleavage with Factor Xa. This novel capture and enzyme-mediated release provided an additional element of selectivity and yielded phosphopeptide-specific modifications with enhanced MS ionization characteristics. The eluted peptides were mapped using MALDI-TOF MS and QTRAP ESI-MS/MS techniques. The results obtained for a model peptide and two tryptic protein digests show that the method is highly specific and allows selective enrichment of phosphorylated peptides at low concentrations of femtomoles per microliter.

Original languageEnglish (US)
Pages (from-to)1007-1017
Number of pages11
JournalJournal of the American Society for Mass Spectrometry
Volume18
Issue number6
DOIs
StatePublished - Jun 1 2007

ASJC Scopus subject areas

  • Structural Biology
  • Spectroscopy

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