Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China.
Anal Chim Acta. 2010 Feb 19;661(1):67-75. doi: 10.1016/j.aca.2009.12.012. Epub 2009 Dec 16.
It is assumed that much more functional importance for protein activity than expected may be granted by methylation that occurs at the side-chain of aspartate or glutamate residue. In vitro methylation mainly comes from the use of methanol in sample preparation prior to MS analysis. In this study, we first performed the methylation site-directed proteomic screening of bovine serum albumin, ovalbumin and 20S proteasome for gel staining using a meaningfully indicative MS-pattern of peak tag (termed as 4P tag) and manual inspection for mass spectral data. As a result, there were 17 proteolytic peptides with 20 modified sites confirmed to be in vitro methylated. Subsequently, the prevalence investigation was performed, focusing on the reaction kinetic behavior of in vitro methylation. This study provided a simple and robust approach for confirmation of in vitro methylation by methanol, as well as the precautious guide for the use of methanol in proteomic study.
据推测，天冬氨酸或谷氨酸侧链发生的甲基化可能赋予蛋白质活性更大的功能重要性。体外甲基化主要来自于在 MS 分析之前使用甲醇进行样品制备。在这项研究中，我们首先使用具有有意义的峰标签（称为 4P 标签）的凝胶染色的 MS 图谱对牛血清白蛋白、卵清蛋白和 20S 蛋白酶体进行了靶向蛋白质组学甲基化筛选，并对质谱数据进行了手动检查。结果，共鉴定出 17 个具有 20 个修饰位点的肽段，这些肽段被确认为体外甲基化。随后，我们进行了流行情况调查，重点研究了体外甲基化的反应动力学行为。该研究提供了一种简单而强大的方法来确认甲醇引起的体外甲基化，以及在蛋白质组学研究中使用甲醇的谨慎指南。
