Capillary Electrophoresis Coupled to Electrospray Ionization Tandem Mass Spectrometry for Ultra-Sensitive Proteomic Analysis of Limited Samples.
Capillary Electrophoresis Coupled to Electrospray Ionization Tandem Mass Spectrometry for Ultra-Sensitive Proteomic Analysis of Limited Samples.
复制标题
毛细管电泳与电喷雾电离串联质谱联用用于有限样本的超灵敏蛋白质组分析
DOI：
10.1021/acs.analchem.1c02929
复制
发表时间：
2022-01-18
影响因子：
7.4
通讯作者：
Ivanov AR
中科院分区：
文献类型：
--
作者：
Johnson KR;Greguš M;Kostas JC;Ivanov AR
在这项工作中，我们开发了一种超灵敏的毛细管电泳 - 串联质谱（CE - MS/MS）方法，用于对有限样本进行自下而上的蛋白质组学分析，可低至总蛋白的亚纳克水平。通过CE - MS/MS对880皮克和88皮克的海拉细胞蛋白消化标准物进行分析，分别鉴定出约1100 ± 46种和约160 ± 59种蛋白质，这表明与目前基于CE - MS/MS且样本量相似的最先进的蛋白质组学分析相比，其蛋白质和肽段的鉴定数量更多。为了展示我们的超灵敏CE - MS/MS方法在分析有限生物样本方面的潜在应用，我们使用小型化的溶液内消化流程对500个和1000个海拉细胞进行消化。从所得消化物中注入相当于1个、5个和10个细胞的量，我们分别鉴定出744 ± 127种、1139 ± 24种和1271 ± 6种蛋白质以及3353 ± 719个、5709 ± 513个和8527 ± 114个肽段组。此外，我们对CE - MS/MS和两种反相纳流液相色谱（RP - nLC - MS/MS）方法（整体柱和填充柱）进行了比较评估，用于分析约10纳克的海拉细胞蛋白消化标准物。我们的结果表明，所评估的基于CE - MS和基于RP - nLC - MS的方法在蛋白质尤其是肽段水平的鉴定上具有互补性。进一步对这些技术进行评估以检测翻译后修饰，并突出了CE - MS/MS方法在鉴定潜在重要且具有生物学相关性的修饰肽段方面的优势。在约60分钟的迁移窗口内，CE - MS/MS从单次注入约8.8纳克的海拉细胞蛋白消化标准物中平均鉴定出约2000 ± 53种蛋白质。此外，在该样本量下，通过CE - MS/MS分析平均鉴定出232 ± 10个磷酸肽段和377 ± 14个N端乙酰化肽段，分别比nLC - MS/MS方法对相应修饰的鉴定数量多2倍和1.5倍。
In this work, we developed an ultra-sensitive CE-MS/MS method for bottom-up proteomics analysis of limited samples, down to sub-nanogram levels of total protein. Analysis of 880 and 88 pg of the HeLa protein digest standard by CE-MS/MS yielded ~1100 ± 46 and ~160 ± 59 proteins, respectively, demonstrating higher protein and peptide identifications than the current state-of-the-art CE-MS/MS-based proteomic analyses with similar amounts of sample. To demonstrate potential applications of our ultra-sensitive CE-MS/MS method for the analysis of limited biological samples, we digested 500 and 1000 HeLa cells using a miniaturized in-solution digestion workflow. From 1-, 5-, and 10-cell equivalents injected from the resulted digests, we identified 744 ± 127, 1139 ± 24, and 1271 ± 6 proteins and 3353 ± 719, 5709 ± 513, and 8527 ± 114 peptide groups, respectively. Furthermore, we performed a comparative assessment of CE-MS/MS and two reversed-phased nano-liquid chromatography (RP-nLC-MS/MS) methods (monolithic and packed columns) for the analysis of a ~10 ng HeLa protein digest standard. Our results demonstrate complementarity in the protein- and especially peptide-level identifications of the evaluated CE-MS- and RP-nLC-MS-based methods. The techniques were further assessed to detect post-translational modifications and highlight the strengths of the CE-MS/MS approach in identifying potentially important and biologically relevant modified peptides. With a migration window of ~60 min, CE-MS/MS identified ~2000 ± 53 proteins on average from a single injection of ~8.8 ng of the HeLa protein digest standard. Additionally, an average of 232 ± 10 phosphopeptides and 377 ± 14 N-terminal acetylated peptides were identified in CE-MS/MS analyses at this sample amount, corresponding to 2- and 1.5-fold more identifications for each respective modification found by nLC-MS/MS methods.
登录
查看更多内容
影响因子：
7.4
作者：
Chen D;Lubeckyj RA;Yang Z;McCool EN;Shen X;Wang Q;Xu T;Sun L
通讯作者：
Sun L
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：
7
作者：
Li, Siyang;Plouffe, Brian D.;Ivanov, Alexander R.
通讯作者：
Ivanov, Alexander R.
DOI：
10.1007/s13361-016-1532-8
发表时间：
2017-04-01
影响因子：
3.2
作者：
Choi, Sam B.;Zamarbide, Marta;Nemes, Peter
通讯作者：
Nemes, Peter
影响因子：
4.1
作者：
Bodor, R;Kaniansky, D;Masár, M
通讯作者：
Masár, M