Wright P John, English Ann M
Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec, Canada H3G 1M8.
J Am Chem Soc. 2003 Jul 16;125(28):8655-65. doi: 10.1021/ja0291888.
The detection and characterization of radicals in biomolecules are challenging due to their high reactivity and low concentration. Mass spectrometry (MS) provides a tool for the unambiguous identification of protein-based radicals by exploiting their reactivity with suitable reagents. To date, protein-radical detection by MS has been modeled after electron paramagnetic resonance experiments, in which diamagnetic spin traps, such as 3,5-dibromo-4-nitrosobenzene sulfonic acid, convert unstable radicals to more stable spin adducts. Since MS detects mass changes, and not unpaired spins, conversion of radicals to stable diamagnetic adducts is more desirable. The use of 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO()) in the MS identification of protein-based radicals was explored here to establish whether scavenging via radical combination would give rise to TEMPO adducts that were stable to MS analysis. The horseradish peroxidase/H(2)O(2) reaction was used to generate radicals in derivatives of tyrosine, tryptophan, and phenylalanine as models of protein-based radicals. TEMPO() was added as a radical scavenger, and the products were analyzed by electrospray ionization (ESI) MS. Dramatically higher mass-adduct yields were obtained using radical scavenging vs radical trapping, which greatly enhanced the sensitivity of radical detection. The efficiency of TEMPO() in protein radical scavenging was examined in horse heart myoglobin and cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae. On H(2)O(2) binding to their ferric hemes, two oxidizing equivalents are transferred to the proteins as an Fe(IV)=O species and a polypeptide-based radical. In addition, CCP has been shown to reduce up to 10 equiv of H(2)O(2) using endogenous donors, thereby generating as many as 20 radicals on its polypeptide. Following myoglobin and CCP incubation with a 10-fold molar excess of H(2)O(2) and TEMPO(), matrix-assisted laser desorption ionization (MALDI) time-of-flight analysis of the tryptic peptides derived from the proteins revealed 1 and 9 TEMPO adducts of myoglobin and CCP, respectively. Given the high scavenging efficiency of TEMPO(*) and the stability of TEMPO-labeled peptides in ESI and MALDI sources, scavenging by stable nitroxide radicals coupled with MS analysis should provide sensitive and powerful technology for the characterization of protein-based radicals.
由于生物分子中的自由基具有高反应活性和低浓度，对其进行检测和表征具有挑战性。质谱（MS）通过利用自由基与合适试剂的反应活性，为明确鉴定基于蛋白质的自由基提供了一种工具。迄今为止，MS检测蛋白质自由基的方法是仿照电子顺磁共振实验建立的，在该实验中，抗磁性自旋捕获剂，如3,5-二溴-4-亚硝基苯磺酸，可将不稳定的自由基转化为更稳定的自旋加合物。由于MS检测的是质量变化而非未成对电子自旋，因此将自由基转化为稳定的抗磁性加合物更为可取。本文探索了使用2,2,6,6-四甲基哌啶-1-氧基（TEMPO()）通过自由基结合清除自由基，从而在MS鉴定基于蛋白质的自由基时，是否会产生对MS分析稳定的TEMPO加合物。以酪氨酸、色氨酸和苯丙氨酸的衍生物作为基于蛋白质的自由基模型，利用辣根过氧化物酶/H₂O₂反应产生自由基。加入TEMPO()作为自由基清除剂，并通过电喷雾电离（ESI）MS对产物进行分析。与自由基捕获相比，自由基清除产生的质量加合物产量显著更高，这大大提高了自由基检测的灵敏度。在马心脏肌红蛋白和酿酒酵母的细胞色素c过氧化物酶（CCP）中检测了TEMPO()清除蛋白质自由基的效率。当H₂O₂与它们的铁血红素结合时，两个氧化当量作为Fe(IV)=O物种和基于多肽的自由基转移到蛋白质上。此外，已证明CCP可利用内源性供体还原多达10当量的H₂O₂，从而在其多肽上产生多达20个自由基。在用10倍摩尔过量的H₂O₂和TEMPO()孵育肌红蛋白和CCP后，对源自这些蛋白质的胰蛋白酶肽段进行基质辅助激光解吸电离（MALDI）飞行时间分析，结果显示肌红蛋白和CCP分别有1个和9个TEMPO加合物。鉴于TEMPO(*)具有较高的清除效率以及TEMPO标记肽段在ESI和MALDI源中的稳定性，通过稳定的氮氧自由基清除结合MS分析应为基于蛋白质的自由基表征提供灵敏且强大的技术。
