One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-μm-i.d. columns increase signal intensity by >3-fold relative to those using 75-μm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos MS significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading to a ~3-fold increase in peptide identifications and 1.7-fold increase in identified protein groups for 2 ng tryptic digests of the bacterium S. oneidensis. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ~ 95% for 0.5 ng samples and by ~42% for 2 ng samples. Using the best combination of the above variables, we were able to identify >3,000 proteins from 10 ng tryptic digests from both HeLa and THP-1 mammalian cell lines. We also identified >950 proteins from subnanogram archaeal/bacterial cocultures. The present ultrasensitive LC-MS platform achieves a level of proteome coverage not previously realized for ultra-small sample loadings, and is expected to facilitate the analysis of subnanogram samples, including single mammalian cells.

中文翻译：

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亚纳克蛋白质组学：LC 色谱柱选择、MS 仪器和数据分析策略对痕量样品蛋白质组覆盖率的影响

基于质谱 (MS) 的蛋白质组学面临的最大挑战之一是分析小样本的能力有限。在这里，我们研究了液相色谱 (LC)、MS 仪器和数据分析方法的相对贡献，目的是提高 0.5 ng 到 50 ng 样品大小的蛋白质组覆盖率。我们表明，使用 30-μm-id 色谱柱的 LC 分离比使用 75-μm-id 色谱柱的信号强度增加了 3 倍以上，从而使肽鉴定增加了 32%。Orbitrap Fusion Lumos MS 相对于上一代 Orbitraps（例如，LTQ-Orbitrap）显着提高了灵敏度和测序速度，导致肽鉴定增加了约 3 倍，2 ng 胰蛋白酶消化物的鉴定蛋白质组增加了 1.7 倍细菌 S. oneidensis。开源 MaxQuant 软件的运行匹配算法进一步将 0.5 ng 样品的蛋白质组覆盖率提高了约 95%，将 2 ng 样品的蛋白质组覆盖率提高了约 42%。使用上述变量的最佳组合，我们能够从来自 HeLa 和 THP-1 哺乳动物细胞系的 10 ng 胰蛋白酶消化物中鉴定 > 3,000 种蛋白质。我们还从亚纳米级古细菌/细菌共培养物中鉴定了 >950 种蛋白质。目前的超灵敏 LC-MS 平台实现了以前超小样品量无法实现的蛋白质组覆盖水平，并有望促进亚纳克样品的分析，包括单个哺乳动物细胞。我们能够从来自 HeLa 和 THP-1 哺乳动物细胞系的 10 ng 胰蛋白酶消化物中鉴定出超过 3,000 种蛋白质。我们还从亚纳米古细菌/细菌共培养物中鉴定了 >950 种蛋白质。目前的超灵敏 LC-MS 平台实现了以前超小样品量无法实现的蛋白质组覆盖水平，并有望促进亚纳克样品的分析，包括单个哺乳动物细胞。我们能够从来自 HeLa 和 THP-1 哺乳动物细胞系的 10 ng 胰蛋白酶消化物中鉴定出超过 3,000 种蛋白质。我们还从亚纳米级古细菌/细菌共培养物中鉴定了 >950 种蛋白质。目前的超灵敏 LC-MS 平台实现了以前超小样品量无法实现的蛋白质组覆盖水平，并有望促进亚纳克样品的分析，包括单个哺乳动物细胞。