Environmental Molecular Sciences Laboratory , Pacific Northwest National Laboratory , Richland , Washington 99354 , United States.
Biological Sciences Division , Pacific Northwest National Laboratory , Richland , Washington 99354 , United States.
Anal Chem. 2019 Aug 6;91(15):9707-9715. doi: 10.1021/acs.analchem.9b01248. Epub 2019 Jul 9.
Two-dimensional reversed-phase capillary liquid chromatography (2D RPLC) separations have enabled comprehensive proteome profiling of biological systems. However, milligram sample quantities of proteins are typically required due to significant losses during offline fractionation. Such a large sample requirement generally precludes the application samples in the nanogram to low-microgram range. To achieve in-depth proteomic analysis of such small-sized samples, we have developed the nanoFAC (nanoflow Fractionation and Automated Concatenation) 2D RPLC platform, in which the first dimension high-pH fractionation was performed on a 75-μm i.d. capillary column at a 300 nL/min flow rate with automated fraction concatenation, instead of on a typically used 2.1 mm column at a 200 μL/min flow rate with manual concatenation. Each fraction was then fully transferred to the second-dimension low-pH nanoLC separation using an autosampler equipped with a custom-machined syringe. We have found that using a polypropylene 96-well plate as collection device as well as the addition of -Dodecyl β-d-maltoside (0.01%) in the collection buffer can significantly improve sample recovery. We have demonstrated the nanoFAC 2D RPLC platform can achieve confident identifications of ∼49,000-94,000 unique peptides, corresponding to ∼6,700-8,300 protein groups using only 100-1000 ng of HeLa tryptic digest (equivalent to ∼500-5,000 cells). Furthermore, by integrating with phosphopeptide enrichment, the nanoFAC 2D RPLC platform can identify ∼20,000 phosphopeptides from 100 μg of MCF-7 cell lysate.
二维反相毛细管液相色谱（2D RPLC）分离技术已经实现了生物系统的全面蛋白质组分析。然而，由于离线分馏过程中的大量损失，通常需要毫克级的蛋白质样品。如此大的样品需求通常排除了纳克到低微克范围的应用样品。为了实现对如此小尺寸样品的深入蛋白质组学分析，我们开发了 nanoFAC（纳米流分馏和自动串联）2D RPLC 平台，其中第一维高 pH 分馏在 75-μm id 的毛细管柱上以 300 nL/min 的流速进行，自动进行馏分串联，而不是在通常使用的 2.1-mm 柱上以 200 μL/min 的流速进行手动串联。然后，使用配备定制加工注射器的自动进样器将每个馏分完全转移到第二维低 pH 纳升 LC 分离中。我们发现，使用聚丙烯 96 孔板作为收集装置以及在收集缓冲液中添加 -Dodecyl β-d-maltoside（0.01%）可以显著提高样品回收率。我们已经证明，nanoFAC 2D RPLC 平台仅使用 100-1000 ng 的 HeLa 酶解物（相当于 500-5,000 个细胞）就可以实现约 49,000-94,000 个独特肽段的可靠鉴定，对应约 6,700-8,300 个蛋白质组。此外，通过与磷酸肽富集相结合，nanoFAC 2D RPLC 平台可以从 100 μg MCF-7 细胞裂解物中鉴定约 20,000 个磷酸肽。
