Department of Chemistry and Chemical Biology, Northeastern University, Barnett Institute of Chemical and Biological Analysis, 360 Huntington Avenue, Boston, Massachusetts 02115, United States.
Anal Chem. 2022 Jan 18;94(2):704-713. doi: 10.1021/acs.analchem.1c02929. Epub 2022 Jan 4.
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.
在这项工作中，我们开发了一种超灵敏的 CE-MS/MS 方法，用于对有限样本进行从头蛋白质组学分析，可达到亚纳克级别的总蛋白水平。通过 CE-MS/MS 对 880 和 88 pg 的 HeLa 蛋白消化标准品进行分析，分别得到约 1100±46 和 160±59 种蛋白质，与使用类似数量的样本的当前最先进的 CE-MS/MS 基于蛋白质组学分析相比，蛋白质和肽的鉴定更多。为了展示我们的超灵敏 CE-MS/MS 方法在分析有限生物样本中的潜在应用，我们使用微型化的溶液内消化工作流程消化了 500 和 1000 个 HeLa 细胞。从注入的 1、5 和 10 个细胞等效物中，我们分别鉴定到 744±127、1139±24 和 1271±6 种蛋白质和 3353±719、5709±513 和 8527±114 种肽段。此外，我们还对 CE-MS/MS 与两种反相纳升液相色谱 (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 ng 的 HeLa 蛋白消化标准品。我们的结果表明，在评估的 CE-MS 和 RP-nLC-MS 基于方法的蛋白质水平，特别是肽水平鉴定方面具有互补性。进一步评估了这些技术以检测翻译后修饰，并突出了 CE-MS/MS 方法在鉴定潜在重要和生物学相关修饰肽方面的优势。CE-MS/MS 在约 60 分钟的迁移窗口内，平均从单次注射约 8.8 ng 的 HeLa 蛋白消化标准品中鉴定到约 2000±53 种蛋白质。此外，在 CE-MS/MS 分析中，在该样品量下，平均鉴定到 232±10 个磷酸肽和 377±14 个 N 端乙酰化肽，分别对应于 nLC-MS/MS 方法鉴定到的每种修饰的 2 倍和 1.5 倍。
