Marley Kevin, Mooney Duane T, Clark-Scannell Gretchen, Tong Tony T-H, Watson Jeffrey, Hagen Tory M, Stevens Jan F, Maier Claudia S
Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA.
J Proteome Res. 2005 Jul-Aug;4(4):1403-12. doi: 10.1021/pr050078k.
A mass tagging approach is described for mitochondrial thiol proteins under nondenaturing conditions. This approach utilizes stable isotope-coded, thiol-reactive (4-iodobutyl)triphenylphosphonium (IBTP) reagents, i.e., the isotopomers IBTP-d(0) and IBTP-d(15). The mass spectrometric properties of IBTP-labeled peptides were evaluated using an ESI-q-TOF and a MALDI-TOF/TOF instrument. High energy collision induced dissociation (CID) in the TOF/TOF instrument caused side-chain fragmentation in the butyltriphenylphosphonium moiety-containing Cys-residue. By contrast, low energy CID in the qTOF instrument yielded sequence tags of IBTP-labeled peptides that were suitable for automated database searching. The IBTP labeling strategy was then applied to the analysis of a protein extract obtained from cardiac mitochondria. The relative abundance measurements for identified IBTP-labeled peptides showed an average variability for peptide quantitation of approximately 10% based on peak area ratios of ion signals for the d(0)/d(15)-tagged peptide pairs. The reactivity of the IBTP reagents was further studied by molecular modeling and visualization. The present study suggests that the IBTP reagent seems to show a bias toward highly surface-exposed protein thiols. Hence, the described mass tagging approach might become potentially useful in redox proteomics studies designed to identify protein thiols that are particularly prone to oxidative modifications.
本文描述了一种在非变性条件下对线粒体硫醇蛋白进行大规模标记的方法。该方法利用稳定同位素编码的硫醇反应性（4-碘丁基）三苯基鏻（IBTP）试剂，即同位素异构体IBTP-d(0)和IBTP-d(15)。使用电喷雾四级杆飞行时间质谱仪（ESI-q-TOF）和基质辅助激光解吸电离飞行时间串联质谱仪（MALDI-TOF/TOF）评估了IBTP标记肽段的质谱特性。在TOF/TOF仪器中进行的高能碰撞诱导解离（CID）导致含丁基三苯基鏻部分的半胱氨酸残基发生侧链断裂。相比之下，qTOF仪器中的低能CID产生了适合自动数据库搜索的IBTP标记肽段的序列标签。然后将IBTP标记策略应用于从心脏线粒体获得的蛋白质提取物的分析。基于d(0)/d(15)标记肽对的离子信号峰面积比，鉴定出的IBTP标记肽段的相对丰度测量显示肽定量的平均变异性约为10%。通过分子建模和可视化进一步研究了IBTP试剂的反应性。本研究表明，IBTP试剂似乎对高度暴露于表面的蛋白质硫醇有偏向性。因此，所描述的大规模标记方法可能在旨在鉴定特别容易发生氧化修饰的蛋白质硫醇的氧化还原蛋白质组学研究中具有潜在用途。
