Mass spectrometry in demonstrating the site-specific nitration of hen egg white lysozyme by an improved electrochemical method
Mass spectrometry in demonstrating the site-specific nitration of hen egg white lysozyme by an improved electrochemical method
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DOI：
10.1016/j.ab.2006.06.033
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发表时间：
2006-09-15
影响因子：
2.9
通讯作者：
Peterson, Ian
中科院分区：
文献类型：
--
作者：
Matters, Dominic;Cooper, Helen J.;Peterson, Ian
在建立一种选择性蛋白质硝化的方法中，我们以前证明了蛋清溶菌酶的电化学硝化最初在Tyr23，然后在Tyr20重硝化，但在Tyr53没有三硝化。通过对包括Tyr23和Tyr20的胰蛋白酶多肽进行测序，确定了硝化位点，但尚未确定对蛋白质其他区域的可能影响。此外，电氧化条件苛刻，氧化电位为+1.2V(与饱和甘汞电极[SCE]相比)，除溶菌酶本身外不添加氮源，与铜旗电极的反应时间较长。在这里，我们报告了一种更温和的方法，使用更短的反应时间，以亚硝酸盐为硝化源，较低的电位(+0.85V vs.SCE)，和铂篮状电极。用电喷雾傅里叶变换离子回旋共振质谱仪对完整蛋白质进行分析，鉴定出单硝化和重硝化产物的质量分别增加了+45和+90 Da，这与NO2取代H的结果一致。此外，结果还表明，电氧化后蛋白质中没有发生其他共价变化。完整的单硝化物种的喷嘴撇油器解离将修饰位点定位于Tyr20或Tyr23。基质辅助激光解吸/电离飞行时间和电喷雾电离飞行时间分析确定单硝化溶菌酶的胰蛋白酶多肽的硝化位置为Tyr23。(C)2006 Elsevier Inc.保留所有权利。
In producing a method for selective protein nitration, we previously demonstrated the electrochemical nitration of hen egg white lysozyme to be at Tyr23 initially, followed by bisnitration at Tyr20, but with no trisnitration at Tyr53. The nitration site was determined by sequencing a tryptic peptide that included Tyr23 and Tyr20, but possible effects on other regions of the protein were not determined. Moreover, the electrooxidation conditions were harsh, involving an oxidation potential of +1.2 V (vs. saturated calomel electrode [SCE]), no added nitrogen source except the lysozyme itself, and long reaction periods with copper flag electrodes. Here we report a gentler procedure using much shorter reaction times with nitrite as the nitration source, a lower potential (+0.85 V vs. SCE), and a platinum basket electrode. Intact protein analysis by electrospray Fourier transform ion cyclotron resonance mass spectrometry identified mono- and bisnitration products with mass increases of +45 and +90 Da, respectively, consistent with the substitution of NO2 for H. In addition, the results revealed that no other covalent change in the protein occurred following electrooxidation. Nozzle skimmer dissociation of the intact mononitrated species localized the modification site to Tyr20 or Tyr23. Matrix-assisted laser desorption/ ionization time-of-flight and electrospray ionization time-of-flight analysis of the tryptic peptides of mononitrated lysozyme identified the site of nitration as Tyr23. (c) 2006 Elsevier Inc. All rights reserved.