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.
KeywordsDehalococcoides mccartyi CBDB1 Absolute protein quantification Selected reaction monitoring Precursor reaction monitoring Organohalide respiration Reductive dehalogenase
- 5.Barr JR, Maggio VL, Patterson DG, Cooper GR, Henderson LO, Turner WE, Smith SJ, Hannon WH, Needham LL, Sampson EJ (1996) Isotope dilution–mass spectrometric quantification of specific proteins: model application with apolipoprotein A-I. Clin Chem 42:1676–1682Google Scholar
- 6.Addona TA, Abbatiello SE, Schilling B, Skates SJ, Mani DR, Bunk DM, Spiegelman CH, Zimmerman LJ, Ham AJ, Keshishian H, Hall SC, Allen S, Blackman RK, Borchers CH, Buck C, Cardasis HL, Cusack MP, Dodder NG, Gibson BW, Held JM, Hiltke T, Jackson A, Johansen EB, Kinsinger CR, Li J, Mesri M, Neubert TA, Niles RK, Pulsipher TC, Ransohoff D, Rodriguez H, Rudnick PA, Smith D, Tabb DL, Tegeler TJ, Variyath AM, Vega-Montoto LJ, Wahlander A, Waldemarson S, Wang M, Whiteaker JR, Zhao L, Anderson NL, Fisher SJ, Liebler DC, Paulovich AG, Regnier FE, Tempst P, Carr SA (2009) Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Nat Biotechnol 27:633–641CrossRefGoogle Scholar
- 17.E. Marco-Urrea, I. Nijenhuis, and L. Adrian (2011) Transformation and carbon iIsotope fractionation of tetra- and trichloroethene to trans-dichloroethene by Dehalococcoides sp. strain CBDB1. Environ Sci Technol. 15(4):1555−1562Google Scholar
- 20.Wagner A, Adrian L, Kleinsteuber S, Andreesen JR, Lechner U (2009) Transcription analysis of genes encoding homologues of reductive dehalogenases in “Dehalococcoides” sp. strain CBDB1 by using terminal restriction fragment length polymorphism and quantitative PCR. Appl Environ Microbiol 75:1876–1884CrossRefGoogle Scholar
- 34.Sonnhammer ELL, von Heijne G, Krogh A (1998) A hidden Markov model for predicting transmembrane helices in protein. Proc Int Conf Intell Syst Mol Biol 6:75–82Google Scholar