Technological Innovation and Resources
MS Western, a Method of Multiplexed Absolute Protein Quantification is a Practical Alternative to Western Blotting*

https://doi.org/10.1074/mcp.O117.067082Get rights and content
Under a Creative Commons license
open access

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.

Cited by (0)

*

Work in AS and NLV laboratories was supported by MPG core funding. AS laboratory was supported by KFO249 and TRR83 (Project A17) grants from Deutsche Forschungsgemeinschaft; NLV laboratory by Human Frontier Science Program Career Development Award (CDA00060/2012). Work in the FB laboratory was supported by the Excellence Initiative of the German Federal and State Governments (Institutional Strategy, measure ‘support the best ZUK 64‘). MK and SRJ are recipients of IMPRS PhD student fellowship.

This article contains supplemental material.

**

Present address: Center for Molecular and Cellular Bioengineering of TU Dresden, 01307 Dresden, Germany.

‡‡

Present address: IMP Research Institute of Molecular Pathology, 1030 Vienna, Austria.

Author contributions: M.K., M.G., and A.S. designed the study. M.K., A.B., and D.D. designed and produced protein chimeras. M.K. performed MS Western benchmarking and validation experiments and quantified zebrafish histones. S.R.J. and N.L.V. performed experiments with zebrafish embryos. M.A. and F.B. performed KD experiments in HeLa cells. M.K. and A.S. wrote the draft that was jointly revised by all coauthors.