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MS Western, a Method of Multiplexed Absolute Protein Quantification is a Practical Alternative to Western Blotting

ABSTRACT: Develeopment and application of MS Western method. A new GeLC-MS/MS method is developed for the absolute quantification of proteins.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine) Danio Rerio (zebrafish) (brachydanio Rerio) Oryctolagus Cuniculus (rabbit) Saccharomyces Cerevisiae (baker's Yeast) Drosophila Melanogaster (fruit Fly)

SUBMITTER: Mukesh Kumar

LAB HEAD: Andrej Shevchenko

PROVIDER: PXD005654 | Pride | 2017-12-18

REPOSITORIES: pride

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			Action	DRS
	Chimeric Protein CP01_Labelled (F048906).mzid_Chimeric_Protein_CP01_Labelled_(F048906).pride.mgf.gz	Mzid
	Chimeric Protein CP01_Labelled (F048906).pride.mztab.gz	Mztab
	Chimeric Protein CP01_Labelled.pride.mgf.gz	Mgf
	Chimeric Protein CP02_Labelled (F048907).mzid_Chimeric_Protein_CP02_Labelled_(F048907).pride.mgf.gz	Mzid
	Chimeric Protein CP02_Labelled (F048907).pride.mztab.gz	Mztab

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MS Western, a Method of Multiplexed Absolute Protein Quantification is a Practical Alternative to Western Blotting.

Kumar Mukesh M Joseph Shai R SR Augsburg Martina M Bogdanova Aliona A Drechsel David D Vastenhouw Nadine L NL Buchholz Frank F Gentzel Marc M Shevchenko Andrej A

Molecular & cellular proteomics : MCP 20171130 2

Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by <i>in-gel</i> codigestion with an isot ...[more]

PMID: 29192002

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MS Western, a Method of Multiplexed Absolute Protein Quantification is a Practical Alternative to Western Blotting.

Project description:Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by in-gel codigestion with an isotopically labeled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance.

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Signaling proteins of a chemosensory pathway from sperm of Arbacia punctulata

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