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

dc.contributor.authorRichard, Vincent R.
dc.contributor.authorDomanski, Dominik
dc.contributor.authorPercy, Andrew J.
dc.contributor.authorBorchers, Christoph H.
dc.date.accessioned2018-02-01T17:22:26Z
dc.date.available2018-02-01T17:22:26Z
dc.date.copyright2017en_US
dc.date.issued2017-07
dc.description.abstractOffline 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.reviewstatusRevieweden_US
dc.description.scholarlevelFacultyen_US
dc.description.sponsorshipThis 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.citationRichard, 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.2017.07.018en_US
dc.identifier.urihttp://dx.doi.org/10.1016/j.jprot.2017.07.018
dc.identifier.urihttp://hdl.handle.net/1828/9031
dc.language.isoenen_US
dc.publisherJournal of Proteomicsen_US
dc.subjectTargeted proteomicsen_US
dc.subjectFractionationen_US
dc.subjectHigh-pH reversed-phase two-dimensionalen_US
dc.subjectchromatographyen_US
dc.subjectMultiple reaction monitoring (MRM)en_US
dc.titleAn online 2D-reversed-phase – Reversed-phase chromatographic method for sensitive and robust plasma protein quantitationen_US
dc.typeArticleen_US

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