Multidimensional separation schemes enhance the identification and molecular characterization of low molecular weight proteomes and short open reading frame encoded peptides in top-down proteomics

In the present work we developed a sample preparation approach for the combined bottom-up and top-down proteomics analysis of small open reading frame encoded proteins (SEP). Key improvements were made by the application of solid phase extraction (SPE) supported enrichment of LMW proteins, followed by two-dimensional LC-MS top-down analysis encompassing both HCD and EThcD ion activation. Bottom-up experiments were used to support and confirm top-down data interpretation. This strategy allowed for the top-down characterisation 36 proteoforms mapping to 12 SEP of the archaea Methanosarcina mazei, and for the first time the identification of posttranslational modifications in these microproteins.

本研究开发了一种适用于小开放阅读框编码蛋白（small open reading frame encoded proteins，SEP）的自下而上与自上而下蛋白质组学联合分析的样品前处理方法。通过固相萃取（solid phase extraction，SPE）辅助富集低分子量蛋白（low molecular weight proteins，LMW）实现关键技术改进，随后采用涵盖高能碰撞解离（high-energy collisional dissociation，HCD）与电子转移诱导高能碰撞解离（electron transfer/higher-energy collisional dissociation，EThcD）两种离子活化模式的二维液相色谱-质谱联用技术开展自上而下分析。本研究借助自下而上实验辅助并验证自上而下的数据解析流程，该策略成功实现了对古菌甲烷八叠球菌（Methanosarcina mazei）12种SEP对应的36种蛋白亚型（proteoforms）的自上而下表征，并首次鉴定了这类微蛋白中的翻译后修饰（posttranslational modifications，PTMs）。