title: Proteomic studies of Syk-interacting proteins using a novel amine-specific isotope tag and GFP nanotrap.
abstract: Green fluorescent protein (GFP) and variants have become powerful tools to study protein localization, interactions, and dynamics. We present here a mass spectrometry-based proteomics strategy to examine protein-protein interactions using anti-GFP single-chain antibody V(H)H in a combination with a novel stable isotopic labeling reagent, isotope tag on amino groups (iTAG). We demonstrate that the single-chain V(H)H (GFP nanotrap) allows us to identify interacting partners of the Syk protein-tyrosine kinase bearing a GFP epitope tag with high efficiency and high specificity. Interacting proteins identified include CrkL, BLNK, α- and β-tubulin, Csk, RanBP5 and DJ-1. The iTAG reagents were prepared with simple procedures and characterized with high accuracy in the determination of peptides in model peptide mixtures and as well as in complex mixture. Applications of the iTAG method and GFP nanotrap to an analysis of the nucleocytoplasmic trafficking of Syk led to the identification of location-specific associations between Syk and multiple proteins. While the results reveal that the new quantitative proteomic strategy is generally applicable to integrate protein interaction data with subcellular localization, extra caution should be taken in evaluating the results obtained by such affinity purification strategies as many interactions appear to occur following cell lysis.
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Jacob A
authors.initials: JA
authors.lastname: Galan
authors.forename: Leela L
authors.initials: LL
authors.lastname: Paris
authors.forename: Hua-jie
authors.initials: HJ
authors.lastname: Zhang
authors.forename: Jacob
authors.initials: J
authors.lastname: Adler
authors.forename: Robert L
authors.initials: RL
authors.lastname: Geahlen
authors.forename: W Andy
authors.initials: WA
authors.lastname: Tao
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002645:Chickens; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000378:metabolism*; D000072377:Syk Kinase; D053719:Tandem Mass Spectrometry
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D057127:Single-Chain Antibodies; D049452:Green Fluorescent Proteins; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase