ZFIN ID: ZDB-PUB-171204-34
MS Western, a method of multiplexed absolute protein quantification is a practical alternative to Western blotting
Kumar, M., Joseph, S.R., Augsburg, M., Bogdanova, A., Drechsel, D., Vastenhouw, N.L., Buchholz, F., Gentzel, M., Shevchenko, A.
Date: 2017
Source: Molecular & cellular proteomics : MCP   17(2): 384-396 (Journal)
Registered Authors: Joseph, Shai, Vastenhouw, Nadine
Keywords: Absolute quantification, Electrophoresis, Histones*, Protein Design, Proteolysis*, QconCAT, Quantification, Western blotting
MeSH Terms:
  • Animals
  • Blotting, Western
  • Chromatography, Liquid
  • Embryo, Nonmammalian
  • Escherichia coli
  • HeLa Cells
  • Histones/chemistry
  • Humans
  • Proteins/analysis
  • Proteomics/methods*
  • Tandem Mass Spectrometry
  • Zebrafish
PubMed: 29192002 Full text @ Mol. Cell. Proteomics
ABSTRACT
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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