Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.
Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.
Anal Bioanal Chem. 2019 Aug;411(21):5363-5372. doi: 10.1007/s00216-018-1452-5. Epub 2018 Nov 5.
Mass spectrometry (MS)-based analysis of complex biological samples is essential for biomedical research and clinical diagnostics. The separation prior to MS plays a key role in the overall analysis, with separations having larger peak capacities often leading to more identified species and improved confidence in those identifications. High-resolution ion mobility (IM) separations enabled by Structures for Lossless Ion Manipulation (SLIM) can provide extremely rapid, high-resolution separations and are well suited as a second dimension of separation following nanoscale liquid chromatography (nanoLC). However, existing sample handling approaches for offline coupling of separation modes require microliter-fraction volumes and are thus not well suited for analysis of trace biological samples. We have developed a novel nanowell-mediated fractionation system that enables nanoLC-separated samples to be efficiently preconcentrated and directly infused at nanoelectrospray flow rates for downstream analysis. When coupled with SLIM IM-MS, the platform enables rapid and high-peak-capacity multidimensional separations of small biological samples. In this study, peptides eluting from a 100 nL/min nanoLC separation were fractionated into ~ 60 nanowells on a microfluidic glass chip using an in-house-developed robotic system. The dried samples on the chip were individually reconstituted and ionized by nanoelectrospray for SLIM IM-MS analysis. Using model peptides for characterization of the nanowell platform, we found that at least 80% of the peptide components of the fractionated samples were recovered from the nanowells, providing up to ~tenfold preconcentration for SLIM IM-MS analysis. The combined LC-SLIM IM separation peak capacities exceeded 3600 with a measurement throughput that is similar to current one-dimensional (1D) LC-MS proteomic analyses. Graphical abstract A nanowell-mediated multidimensional separation platform that combines nanoLC with SLIM IM-MS enables rapid, high-peak-capacity proteomic analyses.
基于质谱（MS）的复杂生物样本分析对于生物医学研究和临床诊断至关重要。MS 之前的分离在整体分析中起着关键作用，具有较大峰容量的分离通常会导致更多被鉴定的物种，并提高这些鉴定的可信度。结构无损离子迁移（SLIM）实现的高分辨率离子迁移（IM）分离可以提供极其快速、高分辨率的分离，非常适合作为纳米液相色谱（nanoLC）之后的第二维分离。然而，现有的用于离线分离模式耦合的样品处理方法需要微升级分体积，因此不适合痕量生物样品的分析。我们开发了一种新颖的纳米孔介导的分馏系统，该系统能够有效地浓缩 nanoLC 分离的样品，并以纳升电喷雾流速直接注入进行下游分析。当与 SLIM IM-MS 耦合时，该平台能够快速、高容量地多维分离小生物样品。在这项研究中，从 100nL/min nanoLC 分离中洗脱的肽使用内部开发的机器人系统在微流控玻璃芯片上分馏成约 60 个纳米孔。芯片上的干燥样品通过纳升电喷雾单独复溶和离子化，用于 SLIM IM-MS 分析。使用模型肽对纳米孔平台进行特征描述，我们发现至少 80%的分馏样品的肽成分从纳米孔中回收，为 SLIM IM-MS 分析提供了高达约十倍的预浓缩。LC-SLIM IM 联合分离的峰容量超过 3600，与当前的一维（1D）LC-MS 蛋白质组学分析的测量通量相似。
