Abstract
This study reports the comprehensive comparison of (15)N metabolic labeling and label free proteomic strategies for quantitation, with particular focus on plant proteomics. Our investigation of proteome coverage, dynamic range and quantitative precision for a wide range of mixing ratios and protein loadings aim to aid the investigators in the decision making process during experimental design. One of the main characteristics of the label free strategy is the applicability to all starting material, which is a limitation to the metabolic labeling. However, particularly at mixing ratios up to 10-fold the (15)N metabolic labeling proved to be more precise. Contrary to usual practice based on the results from this study, we suggest that nonequal mixing ratios in metabolic labeling could further increase the proteome coverage for quantitation. On the other hand, the label free strategy, in combination with low protein loading allows the extension of the dynamic range for quantitation and it is more precise at very high ratios, which could be important for certain types of experiments.
Highlights
Quantitative comparative analyses of proteomes and their dynamic changes under various growth conditions and stimuli has become a widely used approach in experimental and systems biology
Our findings indicate that both methods under comparison, label-free quantitation and 15N metabolic labeling have a number of advantages under certain experimental conditions and constraints
With this study we provide information helping investigators in deciding on the optimal experimental design for their purposes and for making optimal use of the method of choice by considering three major factors influencing quantitation quality, namely proteome coverage, dynamic range and quantitative precision
Summary
Quantitative comparative analyses of proteomes and their dynamic changes under various growth conditions and stimuli has become a widely used approach in experimental and systems biology. In order to compensate for this deficiency the experimenter will have to use different quantitation strategies (e.g. label-free or chemical labeling), or spike in common isotope-labeled reference tissue [23, 24] Despite this vast choice of analytical methods for quantitative proteome profiling, the performance quality regarding proteome coverage, quantitative precision and dynamic range has not been systematically evaluated for plant-derived protein extracts on modern high mass accuracy instruments. In order to provide a solid basis for decision on quantitative strategies in plant proteomics, we performed an in-depth comparison between the metabolic 15N labeling and the label free methods using conditions of wide range of protein amounts per sample and comparative ratios of complex protein mixtures
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