Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM)
Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM)
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DOI：
10.1007/s00216-013-7451-7
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发表时间：
2014-01-01
影响因子：
4.3
通讯作者：
Seifert, Jana
中科院分区：
文献类型：
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作者：
Schiffmann, Christian;Hansen, Rasmus;Seifert, Jana
目标绝对蛋白定量产生有关生物体生理适应的有价值的信息，因此具有很高的兴趣。特别是在这方面，两种基于蛋白质组质谱的技术即选择性反应监测（SRM）和前体反应监测（PRM）被广泛应用。本研究的目的是建立一种最佳的定量分析方法，重点研究严格厌氧细菌Dehalococcoides mccartyi菌株CBDB1中涉及清洁功能的蛋白质和推测的还原脱卤酶蛋白质。这种微生物很小，生长缓慢；因此，它为全面的蛋白质组学分析提供了很少的生物量。因此，我们比较了SRM和PRM技术。两种方法均成功定量了11个多肽。另外，6个肽段分别通过SRM和PRM被单独定量。将多肽加入大肠杆菌裂解液的背景液中，大多数多肽在两种方法下均可在柱上定量至500 amol。在CBDB1裂解液中，用SRM法和PRM法分别检测了15条和14条多肽。对五种脱卤酶的定量分析显示，每个细胞的拷贝数< 10到115个蛋白质分子，这表明在六氯苯上生长时，RdhA蛋白的丰度存在明显差异。我们的结果表明，两种方法具有相当的灵敏度，并且质谱分析的组合导致更高的肽覆盖率，从而更可靠的蛋白质定量。
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.