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J Proteomics. 2017 Sep 25;168:28-36. doi: 10.1016/j.jprot.2017.07.018. Epub 2017 Jul 28.

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

Author information

1
Jewish General Hospital Proteomics Laboratory, McGill University, Lady Davis Institute, 3755 Chemin de la Côte-Sainte-Catherine, Montréal, QC H3T 1E2, Canada.
2
University of Victoria-Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101-4464 Markham St., Victoria, BC V8Z 7X8, Canada.
3
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; Dept of Biochemistry and Microbiology, Petch Building Room 207, 3800 Finnerty Rd., University of Victoria, Victoria, BC V8Z 7X8, Canada; Jewish General Hospital Proteomics Centre, McGill University, 3755 Cote-Ste-Catherine Road, Montreal, QC H3T 1E2, Canada; Department of Oncology, Jewish General Hospital Proteomics Centre, McGill University, 3755 Cote-Ste-Catherine Road, Montreal, QC H3T 1E2, Canada. Electronic address: christoph@proteincentre.com.

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 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.

KEYWORDS:

Fractionation; High-pH reversed-phase two-dimensional chromatography; Multiple reaction monitoring (MRM); Targeted proteomics

PMID:
28757464
DOI:
10.1016/j.jprot.2017.07.018
[Indexed for MEDLINE]
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