Mirzaei Hamid, Regnier Fred
Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
J Chromatogr A. 2006 Nov 17;1134(1-2):122-33. doi: 10.1016/j.chroma.2006.08.096. Epub 2006 Sep 22.
Protein carbonyls are one of the most widely studied markers of oxidative stress. Determining increases in the concentration of protein carbonyls known to be associated with neurodegenerative diseases, heart disease, cancer and ageing. Identification of carbonylation sites in oxidized proteins has been a challenge. Even though recent advances in proteomics has facilitate the identification of carbonylation sites in oxidized proteins, confident identification remains a challenge due to the complicated nature of oxidative damage and the wide range of oxidative modifications. Here, we report the development of a multiplexing strategy that facilitates confident carbonylated peptide identification through a combination of heavy and light isotope coding and a multi-step filtering process. This procedure involves (1) labeling aliquots of oxidized proteins with heavy and light forms of Girard's reagent P (GPR) and combining them in a 1:1 ratio along with (2) LC/MS and MALDI-MS/MS analysis. The filtering process uses LC/MS and MALDI-MS/MS data to rule out false positives by rejecting peptide doublets that do not appear with the correct concentration ratio, retention time, tag number, or resolution. This strategy was used for the identification of heavily oxidized transferrin peptides and resulted in identification 13 distinct peptides. The competency of the method was validated in a complex mixture using oxidized transferrin in a yeast lysate as well as oxidized yeast. Twenty-five percent of the peptides identified in a pure oxidized sample of transferrin were successfully identified from the complex mixture. Analysis of yeast proteome stressed with hydrogen peroxide using this multiplexing strategy resulted in identification of 41 carbonylated peptides from 36 distinct proteins. Differential isotope coding of model peptides at different concentrations followed by mixing at different ratios was used to establish the linear dynamic range for quantification of carbonylated peptides using light and heavy forms of GPR.
蛋白质羰基是研究最为广泛的氧化应激标志物之一。确定已知与神经退行性疾病、心脏病、癌症和衰老相关的蛋白质羰基浓度的增加情况。氧化蛋白质中羰基化位点的鉴定一直是一项挑战。尽管蛋白质组学的最新进展有助于鉴定氧化蛋白质中的羰基化位点，但由于氧化损伤的复杂性和广泛的氧化修饰，可靠的鉴定仍然是一个挑战。在此，我们报告了一种多重分析策略的开发，该策略通过重同位素和轻同位素编码以及多步过滤过程相结合，有助于可靠地鉴定羰基化肽段。该过程包括：（1）用重、轻两种形式的吉拉德试剂P（GPR）标记氧化蛋白质的等分试样，并将它们按1:1的比例混合，以及（2）进行液相色谱/质谱（LC/MS）和基质辅助激光解吸电离质谱/质谱（MALDI-MS/MS）分析。过滤过程利用LC/MS和MALDI-MS/MS数据，通过排除浓度比、保留时间、标签数量或分辨率不正确的肽段双峰来排除假阳性。该策略用于鉴定高度氧化的转铁蛋白肽段，结果鉴定出13种不同的肽段。该方法的能力在使用酵母裂解物中的氧化转铁蛋白以及氧化酵母的复杂混合物中得到了验证。在转铁蛋白的纯氧化样品中鉴定出的肽段，有25%在复杂混合物中成功鉴定出来。使用这种多重分析策略对用过氧化氢处理的酵母蛋白质组进行分析，结果从36种不同的蛋白质中鉴定出41种羰基化肽段。对不同浓度的模型肽段进行差异同位素编码，然后以不同比例混合，用于建立使用轻、重两种形式的GPR对羰基化肽段进行定量的线性动态范围。
