Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine , Christian-Albrechts-Universität zu Kiel , 24105 Kiel , Germany.
J Proteome Res. 2019 Apr 5;18(4):1725-1734. doi: 10.1021/acs.jproteome.8b00948. Epub 2019 Feb 27.
The identification of small proteins and peptides (below ca. 100-150 amino acids) in complex biological samples is hampered by the dominance of higher-molecular-weight proteins. On the contrary, the increasing knowledge about alternative or short open reading frames creates a need for methods that allow the existence of the corresponding gene products to be proven in proteomics experiments. We present an acetonitrile-based precipitation methodology that depletes the majority of proteins above ca. 15 kDa. Parameters such as depletion mixture composition, pH, and temperature were optimized using a model protein mixture, and the method was evaluated in comparison with the established differential solubility method. The approach was applied to the analysis of the low-molecular-weight proteome of the archaea Methanosarcina mazei by means of LC-MS. The data clearly show a beneficial effect from a reduction of complexity, especially in terms of the quality of MS/MS-based identification of small proteins. This fast, detergent-free method allowed for, with minimal sample manipulation, the successful identification of several not yet identified short open reading frame encoded peptides in M. mazei.
在复杂的生物样本中鉴定小蛋白质和肽（低于约 100-150 个氨基酸）受到高分子量蛋白质的主导地位的阻碍。相反，关于替代或短开放阅读框的知识不断增加，这就需要有方法能够在蛋白质组学实验中证明相应基因产物的存在。我们提出了一种基于乙腈的沉淀方法，该方法可以去除大部分分子量大于约 15 kDa 的蛋白质。使用模型蛋白质混合物优化了沉淀混合物组成、pH 值和温度等参数，并将该方法与已建立的差异溶解度方法进行了比较。该方法应用于古菌甲烷八叠球菌的低分子量蛋白质组学的 LC-MS 分析。数据清楚地显示出减少复杂性的有益效果，特别是在基于 MS/MS 的小蛋白质鉴定的质量方面。这种快速、无去污剂的方法可以最小化样品处理，成功鉴定了甲烷八叠球菌中几个尚未鉴定的短开放阅读框编码肽。
