Dataset Information

Macro-porous Reversed Phase Separation of Proteins Combined with Reversed Phase Separation of Phosphopeptides and Tandem Mass Spectrometry for Profiling the Phosphoproteome of MDA-MB-231 cells

ABSTRACT: A new method of combining macro-porous reversed phase (mRP) protein fractionation with reversed phase liquid chromatography (RPLC) peptide separation tandem mass spectrometry (MS/MS) is reported for profiling the phosphoproteome of a complex sample. In this method, an mRP-C18 column was used to fractionate the proteins from a whole cell lysate of a breast cancer cell line, MDA-MB-231, into 38 fractions. Each fraction was subjected to tryptic digestion, sequential phosphopeptide enrichment by immobilized metal ion affinity chromatography (IMAC) and titanium dioxide (TiO2), followed by capillary RPLC-MS/MS analysis. For comparison, the conventional method of using strong cation exchange (SCX)-RPLC separation of peptides combined with MS/MS was also used for analyzing the phosphoproteome. Replicate experiments by the mRP-RPLC method identified 1585 distinct phosphoproteins with 4519 phosphopeptides, compared to 1585 phosphoproteins with 4297 phosphopeptides by SCX-RPLC, with a total of 1947 phosphoproteins and 6278 phosphopeptides identified from the combined results. While the two methods have similar ability in the identification of the phosphoproteome, they produce complementary information. The phosphoproteins identified in this study, including 67 novel phosphorylation sites from 56 breast cancer related proteins, can serve as the entry point for future validation with biological implications in breast cancer.

INSTRUMENT(S): Waters instrument model

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Breast Cancer

SUBMITTER: Xiaoxia Ye

LAB HEAD: Xiaoxia Ye

PROVIDER: PXD000948 | Pride | 2014-07-24

REPOSITORIES: Pride

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Publications

Macroporous reversed-phase separation of proteins combined with reversed-phase separation of phosphopeptides and tandem mass spectrometry for profiling the phosphoproteome of MDA-MB-231 cells.

Ye Xiaoxia X Li Liang L

Electrophoresis 20140728 24

A new method of combining macroporous RP (mRP) protein fractionation with RPLC peptide separation MS/MS is reported for profiling the phosphoproteome of a complex sample. In this method, an mRP-C18 column was used to fractionate the proteins from a whole cell lysate of a breast cancer cell line, MDA-MB-231, into 38 fractions. Each fraction was subjected to tryptic digestion, sequential phosphopeptide enrichment by immobilized metal ion affinity chromatography and titanium dioxide (TiO2 ), follow ...[more]

PMID: 24888630

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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

Macro-porous Reversed Phase Separation of Proteins Combined with Reversed Phase Separation of Phosphopeptides and Tandem Mass Spectrometry for Profiling the Phosphoproteome of MDA-MB-231 cells

Publications

Macroporous reversed-phase separation of proteins combined with reversed-phase separation of phosphopeptides and tandem mass spectrometry for profiling the phosphoproteome of MDA-MB-231 cells.

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