An online 2D-reversed-phase – Reversed-phase chromatographic method for sensitive and robust plasma protein quantitation

Publication date: Available online 28 July 2017
Source:Journal of Proteomics
Author(s): Vincent R. Richard, Dominik Domanski, Andrew J. Percy, Christoph H. Borchers
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 LC-MRM (2D RPRP) 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 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.

Graphical abstract

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