Hentschel Petra, Krucker Manfred, Grynbaum Marc David, Putzbach Karsten, Bischoff Rainer, Albert Klaus
Institute of Organic Chemistry, University of Tübingen, Germany.
Magn Reson Chem. 2005 Sep;43(9):747-54. doi: 10.1002/mrc.1628.
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).
蛋白激酶ZAP-70参与T细胞活化，并与被称为免疫受体酪氨酸活化基序（ITAM）的酪氨酸磷酸化肽序列相互作用。我们研究了包含氨基酸485 - 496（ALGADDSYYTAR）的胰蛋白酶片段中的调节性磷酸化位点。通过使用毛细管HPLC-NMR微探针在600 MHz下对（1）H/（13）C-NMR进行特异性合成和分析，得到了在Y-492和Y-493酪氨酸的一个或两个位点上磷酸化的四种可能的肽。通过磷酸化对芳香族酪氨酸质子的化学位移影响，可以明确区分这些肽。使用微探针和1.5微升的检测体积，对于各种肽中非常少量的样品也能够进行结构解析。对于注射器进样，使用了15微克的分析物（相当于传统5毫米管中约2毫克）。使用1D和2D技术（（1）H，（1）H-COSY-90，（1）H/（13）C-HSQC和（1）H/（13）C-HMBC），在停流模式下从每种肽少于400纳克记录毛细管HPLC-NMR光谱。
