Lim Jihyeon, Vachet Richard W
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Anal Chem. 2003 Mar 1;75(5):1164-72. doi: 10.1021/ac026206v.
Efforts have been made to develop a method that uses metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) to identify the binding site of copper in metalloproteins. This method uses MCO reactions to oxidize the amino acids in the metal-binding site and MS to identify the amino acids that have been oxidized. Several reaction conditions, including Cu(II)/ascorbate/O2, Cu(II)/O2/H2O2, and Cu(II)/ascorbate/O2/H2O2, have been tested at varying concentrations to find the optimum conditions for specific oxidation of only the amino acids bound to copper. For small peptides, such as angiotensin I (Agt I) and [Gln11]-amyloid-beta-protein fragment 1-16 (A beta(1-16)), the optimum conditions for specific modification involve the use of Cu(II)/ascorbate/O2. For a larger protein, azurin, the speed and specificity of the MCO reactions are enhanced by the presence of a relatively high concentration of ascorbate (100 mM) and a small concentration of H2O2 (1 mM). Optimized reaction conditions combined with MS/MS and MSn analysis on a quadrupole ion trap mass spectrometer allow the copper-binding sites to be specifically identified. For Agt I and A beta(1-16), the amino acids bound to copper can be identified without any false positives. For azurin, four of the five amino acids bound to copper are identified with one false positive. This false positive, however, corresponds to the oxidation of Met44, which is probably due to its susceptibility to oxidation and its proximity to the only residue not identified (i.e., Gly45). The results altogether suggest that MCO reactions and MS provide a very promising approach for identifying the amino acid residues bound to copper in metalloproteins.
人们一直在努力开发一种方法，该方法利用金属催化氧化（MCO）反应和质谱（MS）来识别金属蛋白中铜的结合位点。此方法使用MCO反应氧化金属结合位点中的氨基酸，并通过MS识别被氧化的氨基酸。已经在不同浓度下测试了几种反应条件，包括Cu（II）/抗坏血酸盐/O2、Cu（II）/O2/H2O2和Cu（II）/抗坏血酸盐/O2/H2O2，以找到仅特异性氧化与铜结合的氨基酸的最佳条件。对于小肽，如血管紧张素I（Agt I）和[Gln11]-淀粉样β蛋白片段1-16（Aβ(1-16)），特异性修饰的最佳条件涉及使用Cu（II）/抗坏血酸盐/O2。对于较大的蛋白质蓝铜蛋白，相对高浓度的抗坏血酸盐（100 mM）和低浓度的H2O2（1 mM）的存在增强了MCO反应的速度和特异性。在四极杆离子阱质谱仪上结合MS/MS和MSn分析的优化反应条件能够特异性识别铜结合位点。对于Agt I和Aβ(1-16)，可以明确识别与铜结合的氨基酸，没有任何假阳性。对于蓝铜蛋白，五个与铜结合的氨基酸中有四个被识别，有一个假阳性。然而，这个假阳性对应于Met44的氧化，这可能是由于其易氧化以及与唯一未被识别的残基（即Gly45）相邻。总体结果表明，MCO反应和MS为识别金属蛋白中与铜结合的氨基酸残基提供了一种非常有前景的方法。
