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,‡ and Paul Vouros*,† Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, Boston Massachusetts 02115, and Molecular Oncology Research Institute, Tufts-New England Medical Center and Tufts University School of Medicine, Boston Massachusetts 02111 Received January 19, 2007
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. Keywords: phosphorylation • protein • peptide • Tpl2 • IMAC • Pro-Q diamond • cancer • kinase • post-translational modification • capillary LC-nanospray-MS/MS
Introduction Advances in analytical techniques, specifically in mass spectrometry, have allowed for both facile protein identification and routine sequencing of proteins at increased sensitivity levels. Although these improvements are crucial to gain a better understanding of the proteome, it is often post-translational modifications to a protein that prove to be biologically significant as markers of disease. Investigation of protein modification presents additional challenges as these modifications occur at low stoichiometries and often change the analytical behavior of the molecule. For example, the detection of reversible protein phosphorylation is difficult due to its presence at extremely low levels (
