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Absolute Quantitation of In Vitro Expressed Plant Membrane Proteins by Targeted Proteomics (MRM) for the Determination of Kinetic Parameters

  • Carsten Rautengarten
  • Berit Ebert
  • Joshua L. Heazlewood
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1696)

Abstract

The purification of a functional soluble protein from biological or in vitro expression systems can be problematic and the enrichment of a functional membrane protein for biochemical analyses can be a serious technical challenge. Recently we have been characterizing plant endomembrane nucleotide sugar transporters using a yeast expression system. However, rather than enriching these in vitro expressed proteins to homogeneity, we have been conducting biochemical characterization of these transport proteins in yeast microsomal fractions. While this approach has enabled us to estimate a variety of kinetic parameters, the accurate determination of the turnover number of an enzyme-substrate complex (kcat) requires that the catalytic site concentration (amount of protein) in the total reaction volume is known. As a result, we have been employing targeted proteomics (multiple reaction monitoring) with peptide standards and a triple quadrupole mass spectrometer to estimate the absolute amount of protein in a mixed protein microsomal fraction. The following method details the steps required to define the absolute quantitation of an in vitro expressed membrane protein to define complete kinetic parameters.

Key words

Membrane proteins Multiple reaction monitoring Enzyme kinetics 

Notes

Acknowledgments

This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy. J.L.H. and B.E. are supported by ARC Future Fellowships [FT130101165 and FT160100276].

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Carsten Rautengarten
    • 1
  • Berit Ebert
    • 1
    • 2
  • Joshua L. Heazlewood
    • 1
    • 2
  1. 1.School of BioSciencesThe University of MelbourneParkvilleAustralia
  2. 2.Joint BioEnergy InstituteLawrence Berkeley National LaboratoryBerkeleyUSA

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