Stachowiak K, Otlewski J, Polanowski A, Dyckes D F
Department of Chemistry, University of Colorado, Denver 80204.
Pept Res. 1990 May-Jun;3(3):148-54.
The arrangement of the disulfide bridges of Cucurbita maxima trypsin inhibitor, CMTI I, has been confirmed by enzymatic and chemical cleavages of the native protein and analysis of the resulting disulfide-bridged fragments using thermospray liquid chromatography/mass spectrometry. Although the disulfide bridges of CMTI I have recently been assigned from the x-ray crystallographic structure, direct chemical analysis of the S-S bonds using classical techniques proved difficult. The CMTI I molecule is extremely resistant to enzymatic digestion, and only one site of the peptide chain (Met-8) can be used efficiently for chemical cleavage. A series of degradative conditions were employed in the studies reported here. The progress of protein modification was monitored directly by high-performance liquid chromatography/thermospray mass spectrometry. The disulfide pairings could be deduced directly from the mass spectra of the peptides produced by the fragmentation processes and resolved by high-performance liquid chromatography. In two instances, fragments involving a disulfide bond were isolated and further analyzed, and these confirmed the mass spectral assignments. The disulfide bridges identified, 3-20, 10-22 and 16-28, correspond to those of the x-ray structure and are consistent with those assigned for two other closely related trypsin inhibitors.
通过对天然蛋白进行酶解和化学裂解，并使用热喷雾液相色谱/质谱对所得的二硫键连接片段进行分析，证实了南瓜胰蛋白酶抑制剂CMTI I中二硫键的排列方式。尽管最近已根据X射线晶体结构确定了CMTI I的二硫键，但使用经典技术对S-S键进行直接化学分析却很困难。CMTI I分子对酶解具有极强的抗性，肽链上只有一个位点（Met-8）可有效地用于化学裂解。本文报道的研究采用了一系列降解条件。通过高效液相色谱/热喷雾质谱直接监测蛋白质修饰的进程。二硫键配对可直接从片段化过程产生的肽的质谱图中推导出来，并通过高效液相色谱进行解析。在两个实例中，分离出了涉及二硫键的片段并进行了进一步分析，这些结果证实了质谱分析的结果。所确定的二硫键3-20、10-22和16-28与X射线结构中的二硫键相对应，并且与为其他两种密切相关的胰蛋白酶抑制剂所确定的二硫键一致。
