An online 2D-reversed-phase – Reversed-phase chromatographic method for sensitive and robust plasma protein quantitation
dc.contributor.author | Richard, Vincent R. | |
dc.contributor.author | Domanski, Dominik | |
dc.contributor.author | Percy, Andrew J. | |
dc.contributor.author | Borchers, Christoph H. | |
dc.date.accessioned | 2018-02-01T17:22:26Z | |
dc.date.available | 2018-02-01T17:22:26Z | |
dc.date.copyright | 2017 | en_US |
dc.date.issued | 2017-07 | |
dc.description.abstract | 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 severalmanualmanipulations and is prone to sample losses. To address these issues, we developed an online two dimensional high-pH – low-pH reversed-phase-eversedphase (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. | en_US |
dc.description.reviewstatus | Reviewed | en_US |
dc.description.scholarlevel | Faculty | en_US |
dc.description.sponsorship | This work was supported by funding from Genome Canada and Genome British Columbia to the University of Victoria - Genome BC Proteomics Centre (project codes 204PRO for operations and 214PRO for technology development). CHB is grateful for support from the Leading Edge Endowment Fund (LEEF). CHB is also grateful for support from the Segal McGill Chair in Molecular Oncology at McGill University (Montreal, Quebec, Canada), and for support from the Warren Y. Soper Charitable Trust and the Alvin Segal Family Foundation to the Jewish General Hospital (Montreal, Quebec, Canada). | en_US |
dc.identifier.citation | Richard, V.R., Domanski, D., Percy, A.J. & Borchers, C.H. (2017). An online 2Dreversed- phase – Reversed-phase chromatographic method for sensitive and robust plasma protein quantitation. Journal of Proteomics, 168(September), 28-36. http://dx.doi.org/10.1016/j.jprot.2017.07.018 | en_US |
dc.identifier.uri | http://dx.doi.org/10.1016/j.jprot.2017.07.018 | |
dc.identifier.uri | http://hdl.handle.net/1828/9031 | |
dc.language.iso | en | en_US |
dc.publisher | Journal of Proteomics | en_US |
dc.subject | Targeted proteomics | en_US |
dc.subject | Fractionation | en_US |
dc.subject | High-pH reversed-phase two-dimensional | en_US |
dc.subject | chromatography | en_US |
dc.subject | Multiple reaction monitoring (MRM) | en_US |
dc.title | An online 2D-reversed-phase – Reversed-phase chromatographic method for sensitive and robust plasma protein quantitation | en_US |
dc.type | Article | en_US |