Nanosecond laser-induced photochemical oxidation method for protein surface mapping with mass spectrometry
Nanosecond laser-induced photochemical oxidation method for protein surface mapping with mass spectrometry
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DOI：
10.1021/ac050353m
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发表时间：
2005-09-15
影响因子：
7.4
通讯作者：
Sze, SK
中科院分区：
文献类型：
--
作者：
Aye, TT;Low, TY;Sze, SK
我们开发了一种激光诱导蛋白质表面氧化的超快脉冲蛋白质表面足迹方法。这种方法利用脉冲紫外激光，通过光解过氧化氢(H_2O_2)溶液在纳秒内产生高浓度的羟基(OH)自由基。羟基自由基氧化蛋白质表面的氨基酸残基，产生稳定的共价键修饰。然后对氧化的蛋白质进行质谱分析，以绘制氧化的氨基酸残基。本研究以泛素和脱脂肌红蛋白为模型蛋白。我们的结果表明，单个激光脉冲可以产生广泛的蛋白质表面氧化。我们发现，与未氧化泛素相比，单氧化泛素在随后的激光照射下更容易发生进一步的氧化。这是由于氧化使蛋白质的构象发生变化，从而增加了溶剂可及表面积。因此，为了避免蛋白质构象改变后蛋白质的氧化，用单脉冲激光进行这项实验至关重要。随后，为了在保持激光次数不变的情况下获得更高的氧化频率和覆盖范围，我们进一步优化了激光功率、过氧化氢浓度以及蛋白质浓度。这种超快的羟基自由基生成方法可以快速准确地检测表面残基，从而能够绘制蛋白质在其天然状态下的溶剂可及区域。
We have developed an ultrafast pulse method for protein surface footprinting by laser-induced protein surface oxidations. This method makes use of a pulse UV laser that produces, in nanoseconds, a high concentration of hydroxyl (OH) free radicals by photodissociation of a hydrogen peroxide (H2O2) solution. The OH radicals oxidize amino acid residues located on the protein surface to produce stable covalent modifications. The oxidized protein is then analyzed by mass spectrometry to map the oxidized amino acid residues. Ubiquitin and apomyoglobin were used as model proteins in this study. Our results show that a single laser pulse can produce extensive protein surface oxidations. We found that monooxidized ubiquitins were more susceptible to further oxidations by subsequent laser irradiation, as compared to nonoxidized ones. This is due to the conformational changes of proteins by oxidation that increases the solvent-accessible surface area. Therefore, it is crucial to perform this experiment with a single pulse of laser so as to avoid oxidation of proteins after conformation of the protein changes. Subsequently, to obtain a high frequency and coverage of the oxidation sites while keeping the number of laser shots to one, we further optimized the laser power and concentration of hydrogen peroxide as well as the concentration of protein. This ultrafast OH radical generation method allows for rapid and accurate detection of surface residues, enabling mapping of the solvent-accessible regions of a protein in its native state.