Analytical and Bioanalytical Chemistry

, Volume 406, Issue 1, pp 283–291 | Cite as

Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM)

  • Christian Schiffmann
  • Rasmus Hansen
  • Sven Baumann
  • Anja Kublik
  • Per Halkjær Nielsen
  • Lorenz Adrian
  • Martin von Bergen
  • Nico Jehmlich
  • Jana Seifert
Research Paper

Abstract

Targeted absolute protein quantification yields valuable information about physiological adaptation of organisms and is thereby of high interest. Especially for this purpose, two proteomic mass spectrometry-based techniques namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM) are commonly applied. The objective of this study was to establish an optimal quantification assay for proteins with the focus on those involved in housekeeping functions and putative reductive dehalogenase proteins from the strictly anaerobic bacterium Dehalococcoides mccartyi strain CBDB1. This microbe is small and slow-growing; hence, it provides little biomass for comprehensive proteomic analysis. We therefore compared SRM and PRM techniques. Eleven peptides were successfully quantified by both methods. In addition, six peptides were solely quantified by SRM and four by PRM, respectively. Peptides were spiked into a background of Escherichia coli lysate and the majority of peptides were quantifiable down to 500 amol absolute on column by both methods. Peptide quantification in CBDB1 lysate resulted in the detection of 15 peptides using SRM and 14 peptides with the PRM assay. Resulting quantification of five dehalogenases revealed copy numbers of <10 to 115 protein molecules per cell indicating clear differences in abundance of RdhA proteins during growth on hexachlorobenzene. Our results indicated that both methods show comparable sensitivity and that the combination of the mass spectrometry assays resulted in higher peptide coverage and thus more reliable protein quantification.

Figure

Dehalococcoides mccartyi strain CBDB1 cultivated on hexachlorobenzene were used to compare two targeted peptide quantification assays for reductive dehalogenases, namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM)

Keywords

Dehalococcoides mccartyi CBDB1 Absolute protein quantification Selected reaction monitoring Precursor reaction monitoring Organohalide respiration Reductive dehalogenase 

Supplementary material

216_2013_7451_MOESM1_ESM.pdf (223 kb)
ESM 1(PDF 223 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Christian Schiffmann
    • 1
  • Rasmus Hansen
    • 2
  • Sven Baumann
    • 3
  • Anja Kublik
    • 4
  • Per Halkjær Nielsen
    • 2
  • Lorenz Adrian
    • 4
  • Martin von Bergen
    • 1
    • 2
    • 3
  • Nico Jehmlich
    • 1
  • Jana Seifert
    • 1
    • 5
  1. 1.Department of ProteomicsHelmholtz Centre for Environmental Research—UFZLeipzigGermany
  2. 2.Department of Biotechnology, Chemistry and Environmental EngineeringAalborg UniversityAalborgDenmark
  3. 3.Department of MetabolomicsHelmholtz Centre for Environmental Research—UFZLeipzigGermany
  4. 4.Department Isotope BiogeochemistryHelmholtz Centre for Environmental Research—UFZLeipzigGermany
  5. 5.Institute of Animal NutritionHohenheim UniversityStuttgartGermany

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