title: Determination of regulatory phosphorylation sites in nanogram amounts of a synthetic fragment of ZAP-70 using microprobe NMR and on-line coupled capillary HPLC-NMR.
abstract: The protein kinase ZAP-70 is involved in T-cell activation and interacts with tyrosine-phosphorylated peptide sequences known as immunoreceptor tyrosine activation motifs (ITAMs). We have studied the regulatory phosphorylation sites in the tryptic fragment containing amino acids 485-496 (ALGADDSYYTAR). The four possible peptides with phosphorylation at none, one, or both of the Y-492 and Y-493 tyrosines were specifically synthesized and analyzed by (1)H/(13)C-NMR at 600 MHz using a capillary HPLC-NMR microprobe. Unambiguous discrimination of the peptides was possible via effect of chemical shifts of phosphorylation on the aromatic tyrosine protons. With the microprobe and the detection volume of 1.5 microl, it was possible to perform structure elucidation with the very small amounts available for the various peptides. For the syringe injection, 15 microg of the analyte were used (corresponding to ca 2 mg in classical 5-mm tubes). Capillary HPLC-NMR spectra were recorded in the stopped-flow mode from less than 400 ng of each peptide, using 1D and 2D techniques ((1)H,(1)H-COSY-90, (1)H/(13)C-HSQC, and (1)H/(13)C-HMBC).
authors.affiliation: Institute of Organic Chemistry, University of Tübingen, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Hentschel
authors.forename: Manfred
authors.initials: M
authors.lastname: Krucker
authors.forename: Marc David
authors.initials: MD
authors.lastname: Grynbaum
authors.forename: Karsten
authors.initials: K
authors.lastname: Putzbach
authors.forename: Rainer
authors.initials: R
authors.lastname: Bischoff
authors.forename: Klaus
authors.initials: K
authors.lastname: Albert
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008832:Microchemistry; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase
chemical_list: D000596:Amino Acids; D010455:Peptides; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase