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

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...

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Bibliographic Details
Published in:Analytical and bioanalytical chemistry 2014, Vol.406 (1), p.283-291
Main Authors: Schiffmann, Christian, Hansen, Rasmus, Baumann, Sven, Kublik, Anja, Nielsen, Per Halkjær, Adrian, Lorenz, von Bergen, Martin, Jehmlich, Nico, Seifert, Jana
Format: Article
Language:English
Subjects:
Accuracy
Anaerobiosis
Analytical Chemistry
Assaying
Bacterial Proteins - chemistry
Bacterial Proteins - metabolism
Biochemistry
Biomass
Characterization and Evaluation of Materials
Chemistry
Chemistry and Materials Science
Chloroflexi - chemistry
Chloroflexi - drug effects
Chloroflexi - metabolism
Chromatography, High Pressure Liquid - methods
Escherichia coli - chemistry
Food Science
Genomes
Hexachlorobenzene - metabolism
Hexachlorobenzene - pharmacology
Hydrolases - chemistry
Hydrolases - metabolism
Isoenzymes - chemistry
Isoenzymes - metabolism
Laboratory Medicine
Mass spectrometry
Metabolism
Microorganisms
Monitoring
Monitoring/Environmental Analysis
Oxidation-Reduction
Peptide Fragments - analysis
Peptides
Precursors
Proteins
Proteomics
Proteomics - methods
Reproduction
Research Paper
Respiration
Scientific imaging
Tandem Mass Spectrometry - methods
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Summary: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
ISSN:1618-2642
1618-2650
DOI:10.1007/s00216-013-7451-7