Department of Chemistry, Sapienza Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, Italy.
CNR NANOTEC, Campus Ecotekne, University of Salento, Via Monteroni, 73100 Lecce, Italy.
Anal Chem. 2020 Jun 2;92(11):7964-7971. doi: 10.1021/acs.analchem.0c01342. Epub 2020 May 20.
Protein tyrosine O-sulfation is an important post-translational modification, as it has been correlated to inflammation, virus infection, and signal pathways. Nevertheless, methods for the characterization of protein sulfation by sulfopeptide enrichment are currently limited. In this Article, two standard compounds, representative of mono- and disulfated peptides, were used to compare the enrichment capabilities of five sorbent materials: two commercial weak anion-exchange mixed-mode sorbents (Strata X-AW and Oasis WAX) and three phosphopeptide enrichment materials based on affinity chromatography to either immobilized metals (IMAC) or metal oxides, i.e., Fe, TiO, or Ti. The sulfopeptides were analyzed by ultrahigh-performance liquid chromatography (UHPLC) multiple-reaction monitoring analysis and were stable under all the tested experimental conditions. Recoveries of the enrichment step from spiked bovine serum albumin digests were >80% for the commercial Fe-IMAC kit and the Strata X-AW sorbent. Shotgun proteomics was used on the same samples to evaluate the selectivity, calculated as the number of coenriched peptides, and it was found to be better for the Fe-IMAC kit. Therefore, the Fe-IMAC protocol was embedded in a shotgun-proteomics workflow and applied to serum spiked with the sulfopeptides before protein dephosphorylation and digestion. The recovery of the entire analytical workflow was 20%, which was compatible with previous data on TiO phosphopeptide enrichment. Despite the potential, no sulfopeptide was confidently identified in serum digests by conventional shotgun proteomics, probably due to very low abundance of native sulfoproteins, poor ionization efficiency of sulfopeptides in the positive mode, and lack of unambiguous sulfopeptide identification by bioinformatics software. In this context, the use of negative-ionization mode with high-resolution mass spectrometry appeared promising to improve the sensibility and allow sulfopeptide identification in complex samples.
蛋白质酪氨酸 O-硫酸化是一种重要的翻译后修饰，因为它与炎症、病毒感染和信号通路有关。然而，目前用于通过硫酸肽富集来表征蛋白质硫酸化的方法受到限制。在本文中，使用了两种标准化合物，代表单硫酸化和二硫酸化肽，以比较五种吸附材料的富集能力：两种商业弱阴离子交换混合模式吸附剂（Strata X-AW 和 Oasis WAX）和三种基于亲和色谱的磷酸肽富集材料，分别为固定化金属（IMAC）或金属氧化物，即 Fe、TiO 或 Ti。硫酸肽通过超高效液相色谱（UHPLC）多反应监测分析进行分析，在所有测试的实验条件下均稳定。从牛血清白蛋白消化物中添加的回收率>80%，商业 Fe-IMAC 试剂盒和 Strata X-AW 吸附剂。对相同的样品进行鸟枪法蛋白质组学分析，以评估选择性，计算为共同富集的肽的数量，发现 Fe-IMAC 试剂盒更好。因此，Fe-IMAC 方案被嵌入鸟枪法蛋白质组学工作流程中，并应用于血清中添加硫酸肽后进行蛋白质去磷酸化和消化。整个分析工作流程的回收率为 20%，与 TiO 磷酸肽富集的先前数据兼容。尽管有潜力，但传统鸟枪法蛋白质组学在血清消化物中没有鉴定出任何硫酸肽，可能是由于天然硫酸蛋白丰度非常低、硫酸肽在正模式下的离子化效率差以及生物信息学软件缺乏明确的硫酸肽鉴定。在这种情况下，使用具有高分辨率质谱的负离子模式似乎有望提高灵敏度，并允许在复杂样品中鉴定硫酸肽。
