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 (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.
Department of Proteomics, Helmholtz Centre for Environmental Research-UFZ, Permoserstr. 15, 04318, Leipzig, Germany.
Christian
C
Schiffmann
Rasmus
R
Hansen
Sven
S
Baumann
Anja
A
Kublik
Per Halkjær
PH
Nielsen
Lorenz
L
Adrian
Martin
M
von Bergen
Nico
N
Jehmlich
Jana
J
Seifert
D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D041862:Chloroflexi / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry; D006581:Hexachlorobenzene / Q000378:metabolism / Q000494:pharmacology; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
D001426:Bacterial Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D006581:Hexachlorobenzene; D006867:Hydrolases