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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Dataset's files

Source:

			Action	DRS
	SCX-RPLC_IMAC_Replicate 1_Fraction 1.pride.mgf.gz	Mgf
	SCX-RPLC_IMAC_Replicate 1_Fraction 1.pride.mztab.gz	Mztab
	SCX-RPLC_IMAC_Replicate 1_Fraction 10.pride.mgf.gz	Mgf
	SCX-RPLC_IMAC_Replicate 1_Fraction 10.pride.mztab.gz	Mztab
	SCX-RPLC_IMAC_Replicate 1_Fraction 11.pride.mgf.gz	Mgf

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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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