title: Characterization of phosphorylation sites on Tpl2 using IMAC enrichment and a linear ion trap mass spectrometer.
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.
authors.affiliation: Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical Biology, Northeastern University, Boston Massachusetts 02115, USA.
authors.forename: Terrence M
authors.initials: TM
authors.lastname: Black
authors.forename: Christine L
authors.initials: CL
authors.lastname: Andrews
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Kilili
authors.forename: Mircea
authors.initials: M
authors.lastname: Ivan
authors.forename: Philip N
authors.initials: PN
authors.lastname: Tsichlis
authors.forename: Paul
authors.initials: P
authors.lastname: Vouros
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D007093:Imidazoles / Q000737:chemistry*; D020930:MAP Kinase Kinase Kinases / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000737:chemistry*
chemical_list: D007093:Imidazoles; D010748:Phosphopeptides; D011518:Proto-Oncogene Proteins; D020930:MAP Kinase Kinase Kinases; C069937:MAP3K8 protein, human; C005954:imidazoleacetic acid