Dataset Information

Online non-contiguous fractionating and concatenating device coupled to two dimensional reverse phase/reverse phase liquid chromatography system for efficient and comprehensive proteomic analyses

ABSTRACT: The goal of the project is development of an online two dimensional reverse phase/reverse phase liquid chromatography (2D-RP/RPLC) separation platform, employing an online non-contiguous fractionating and concatenating device (NCFC fractionator) in order to provide highly improve separation orthogonality of the two RPs, resulting in significant increase in peptide identification sensitivity compared with those of the conventional contiguous 2D-RP/RPLC method.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Stomach

SUBMITTER: Dong-Gi Mun

LAB HEAD: Sang-Won Lee

PROVIDER: PXD004710 | Pride | 2016-12-22

REPOSITORIES: Pride

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Publications

Efficient Exploitation of Separation Space in Two-Dimensional Liquid Chromatography System for Comprehensive and Efficient Proteomic Analyses.

Lee Hangyeore H Mun Dong-Gi DG So Jeong Eun JE Bae Jingi J Kim Hokeun H Masselon Christophe C Lee Sang-Won SW

Analytical chemistry 20161111 23

Proteomics aims to achieve complete profiling of the protein content and protein modifications in cells, tissues, and biofluids and to quantitatively determine changes in their abundances. This information serves to elucidate cellular processes and signaling pathways and to identify candidate protein biomarkers and/or therapeutic targets. Analyses must therefore be both comprehensive and efficient. Here, we present a novel online two-dimensional reverse-phase/reverse-phase liquid chromatography ...[more]

PMID: 27801565

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Project description:Two-dimensional (2D) liquid chromatography (LC)-tandem mass spectrometry (MS/MS) are typically employed for deep bottom-up proteomics, and the state-of-the-art 2D-LC-MS/MS has approached over 8,000 protein identifications (IDs) from mammalian cell lines or tissues in 1-3 days of mass spectrometer time. Capillary zone electrophoresis (CZE)-MS/MS has been suggested as an alternative to LC-MS/MS for bottom-up proteomics. CZE-MS/MS and LC-MS/MS are complementary in protein/peptide ID from complex proteome digests because CZE and LC are orthogonal for peptide separation. In addition, the migration time of peptides from CZE-MS can be predicted accurately, which is invaluable for evaluating the confidence of peptide ID from the database search and even guiding the database search. However, the number of protein IDs from complex proteomes using CZE-MS/MS is still much lower than the state of the art using 2D-LC-MS/MS. In this work, for the first time, we established a strong cation exchange (SCX)-reversed phase LC (RPLC)-CZE-MS/MS platform for deep bottom-up proteomics. The platform identified around 8,200 protein groups and 65,000 unique peptides from a mouse brain proteome digest in 70 hours. The data represents the largest bottom-up proteomics dataset using CZE-MS/MS and provides a valuable resource for further improving the tool for prediction of peptide migration time in CZE. The peak capacity of the orthogonal SCX-RPLC-CZE platform was estimated to be around 7,000. SCX-RPLC-CZE-MS/MS produced comparable numbers of protein and peptide IDs with 2D-LC-MS/MS (8,200 vs. 8,900 protein groups, 65,000 vs. 70,000 unique peptides) from the mouse brain proteome digest using comparable instrument time. This is the first time that CZE-MS/MS showed its capability to approach comparable performance to the state-of-the-art 2D-LC-MS/MS for deep proteomic sequencing. SCX-RPLC-CZE-MS/MS and 2D-LC-MS/MS showed good complementarity in protein and peptide IDs and combining those two methods improved the number of protein group and unique peptide IDs by nearly 10% and over 40%, respectively, compared with 2D-LC-MS/MS alone.

Dataset Information

Online non-contiguous fractionating and concatenating device coupled to two dimensional reverse phase/reverse phase liquid chromatography system for efficient and comprehensive proteomic analyses

Publications

Efficient Exploitation of Separation Space in Two-Dimensional Liquid Chromatography System for Comprehensive and Efficient Proteomic Analyses.

Similar Datasets

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