Characterization of phosphorylation sites on Tpl2 using IMAC enrichment and a linear ion trap mass spectrometer

Terrence M. Black, Christine L. Andrews, Geoffrey Kilili, Mircea Ivan, Philip N. Tsichlis, Paul Vouros

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

Advances in analytical techniques, specifically in mass spectrometry, have allowed for both facile protein identification and routine sequencing of proteins at increased sensitivity levels. Protein modifications present additional challenges because they occur at low stoichiometries and often change the analytical behavior of the molecule. For example, characterization of protein phosphorylation provides crucial information to signaling processes that are often associated with disease. Research into protein phosphorylation requires inter-disciplinary co-operation involving multiple investigators with expertise in diverse scientific fields. As such, techniques must be simple, effective, and incorporate multiple checkpoints that confirm the sample contains a phosphorylated protein in order to ensure resources are conserved. In this study, tumor progression locus 2 (Tpl2), which has been implicated in cell cycle regulation and has been shown to play a significant role in critical signal transduction pathways, was transfected into 293T cells, overexpressed and isolated from the cell lysate. Isolated proteins were separated via 1D gel electrophoresis, and their phosphorylation was confirmed using phosphospecific staining. The bands were excised and subjected to tryptic digestion and immobilized metal affinity chromatography (IMAC) prior to analysis by capillary-LC-MS/MS. Three phosphorylation sites were detected on Tpl2. One site had previously been reported in the literature but had not been characterized by mass spectrometric methods until this time; two additional novel sites of phosphorylation were detected.

Original languageEnglish (US)
Pages (from-to)2269-2276
Number of pages8
JournalJournal of Proteome Research
Volume6
Issue number6
DOIs
StatePublished - Jun 1 2007
Externally publishedYes

Keywords

  • Cancer
  • Capillary LC-nanospray-MS/M
  • IMAC
  • Kinase
  • Peptide
  • Phosphorylation
  • Post-translational modification
  • Pro-Q diamond
  • Protein
  • Tpl2

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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