Single-Shot Top-Down Proteomics with Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry for Identification of Nearly 600 Escherichia coli Proteoforms.
Single-Shot Top-Down Proteomics with Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry for Identification of Nearly 600 Escherichia coli Proteoforms.
复制标题
DOI：
10.1021/acs.analchem.7b02532
复制
发表时间：
2017-11-21
影响因子：
7.4
通讯作者：
Sun L
中科院分区：
文献类型：
--
作者：
Lubeckyj RA;McCool EN;Shen X;Kou Q;Liu X;Sun L
毛细管区带电喷雾串联质谱（CZE-ESI-MS/MS）是自上而下蛋白质组学研究的重要平台。然而，由于CZE-MS/MS的低上样容量和窄分离窗口，使用CZE-MS/MS的自上而下的蛋白质组学的规模仍然受到限制。本研究首次系统评价了动态pH连接法用于CZE-MS中完整蛋白质的富集，优化后的动态pH连接CZE-MS/MS的上样量接近1 μL，分离窗口90 min，高峰容量（~280）。该结果代表了CZE自顶向下表征复杂蛋白质组的最大负载容量和最高峰容量。单次CZE-MS/MS鉴定了约2，800种蛋白质谱匹配，近600种蛋白质型和200种来自大肠杆菌蛋白质组的蛋白质，光谱水平错误发现率（FDR）小于1%。在这项工作中确定的蛋白质型的数量是在以前的单次CZE-MS/MS研究的三倍以上。检测到截短、N-末端甲硫氨酸切除、信号肽去除和一些翻译后修饰，包括氧化和乙酰化。
Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as an invaluable platform for top-down proteomics. However, the scale of top-down proteomics using CZE-MS/MS is still limited due to the low loading capacity and narrow separation window of CZE. In this work, for the first time we systematically evaluated the dynamic pH junction method for focusing of intact proteins during CZE-MS. The optimized dynamic pH junction based CZE-MS/MS approached 1-μL loading capacity, 90-min separation window and high peak capacity (~280) for characterization of an Escherichia coli proteome. The results represent the largest loading capacity and the highest peak capacity of CZE for top-down characterization of complex proteomes. Single-shot CZE-MS/MS identified about 2,800 proteoform-spectrum matches, nearly 600 proteoforms, and 200 proteins from the Escherichia coli proteome with spectrum-level false discovery rate (FDR) less than 1%. The number of identified proteoforms in this work is over three times higher than that in previous single-shot CZE-MS/MS studies. Truncations, N-terminal methionine excision, signal peptide removal and some post-translational modifications including oxidation and acetylation were detected.
登录
查看更多内容
影响因子：
7.4
作者：
Busnel, Jean-Marc;Schoenmaker, Bart;Mayboroda, Oleg A.
通讯作者：
Mayboroda, Oleg A.
影响因子：
4.1
作者：
Imami, Koshi;Monton, Maria Rowena N.;Terabe, Shigeru
通讯作者：
Terabe, Shigeru
影响因子：
5.8
作者：
Kou, Qiang;Xun, Likun;Liu, Xiaowen
通讯作者：
Liu, Xiaowen
影响因子：
7.4
作者：
Britz-McKibbin, P;Chen, DDY
通讯作者：
Chen, DDY
影响因子：
7.4
作者：
Moini, Mehdi
通讯作者：
Moini, Mehdi