bioRxiv Biochemistry
bioRxiv Biochemistry
5 years ago

MS Western, a method of multiplexed absolute protein quantification is a practical alternative to western blotting

Buchholz, Kumar, M., Vastenhouw, N. L., Bogdanova, F., A., Gentzel, D., Drechsel, Shevchenko, S. R., Augsburg, Joseph
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 content of dozens of user-selected proteins at the low femtomole level in whole cell or tissue lysates without metabolic or chemical labelling or using specific antibodies. MS Western relies upon GeLC-MS/MS and quantifies proteins by in-gel co-digestion with an isotopically labelled 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.

Publisher URL: http://biorxiv.org/cgi/content/short/156943v1

DOI: 10.1101/156943

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