Jewish General Hospital Proteomics Laboratory, McGill University, Lady Davis Institute, 3755 Chemin de la Côte-Sainte-Catherine, Montréal, QC H3T 1E2, Canada.
University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada.
J Proteomics. 2017 Sep 25;168:28-36. doi: 10.1016/j.jprot.2017.07.018. Epub 2017 Jul 28.
Offline high-pH reversed-phase fractionation is widely used to reduce sample complexity in proteomic workflows. This is due to the semi-orthogonality and high peak resolution of the two separations. Offline 2D fractionation, however, is low throughput and requires several manual manipulations and is prone to sample losses. To address these issues, we developed an online two dimensional high-pH - low-pH reversed-phase-reversed-phase (2D RPRP) LC-MRM method whereby hundreds of peptides can be quantified in a single LC-MS/MS injection. The method allowed the reproducible and sensitive quantitation of a test panel of 367 peptides (168 proteins) from undepleted and non-enriched human plasma. Of these, we were able to detect and quantify 95 peptides (29 proteins) by 2D-RPRP that were not detectable by 1D LC-MRM-MS. Online 2D RPRP resulted in an average increase of roughly 10-fold in sensitivity compared to traditional 1D low-pH separations, while improving reproducibility and sample throughput relative to offline 2D RPRP by factors of 1.7 and 5, respectively, compared to offline 2D RPRP. This paper serves as proof-of-concept of the feasibility and efficacy of online 2D RPRP at analytical flow rates for highly multiplexed targeted proteomic analyses.
离线高 pH 值反相分级广泛用于减少蛋白质组学工作流程中的样品复杂性。这是由于两种分离的半正交性和高峰分辨率。然而，离线 2D 分级处理通量低，需要进行多次手动操作，并且容易造成样品损失。为了解决这些问题，我们开发了一种在线二维高 pH 值-低 pH 值反相-反相（2D RPRP）LC-MRM 方法，通过单次 LC-MS/MS 进样即可定量数百种肽。该方法允许从未耗尽和非富集的人类血浆中重现性和灵敏地定量测试面板中的 367 个肽（168 个蛋白质）。其中，我们能够通过 2D-RPRP 检测和定量 95 个肽（29 个蛋白质），而通过 1D LC-MRM-MS 无法检测到这些肽。与传统的 1D 低 pH 值分离相比，在线 2D RPRP 平均提高了约 10 倍的灵敏度，同时与离线 2D RPRP 相比，重复性和样品通量分别提高了 1.7 倍和 5 倍。本文为在线 2D RPRP 在高多重靶向蛋白质组学分析的分析流速下的可行性和功效提供了概念验证。
