Reproducibility Assessment of Relative Quantitation Strategies for LC−MS Based Proteomics

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Abstract
The reproducibility of a given method for relative quantitation governs the reliability of liquid chromatography−mass spectrometry (LC−MS) based differential analysis in proteomic studies. Understanding the noise level introduced from biological, chemical, and instrumental sources not only helps to determine the experimental design but also aids in assessing the reliability of expression ratios used for quantitation. Here we present a reproducibility assessment method for relative quantitation based on the intensity ratio distribution of common features in LC−MS replicates. This method applies to both decoupled (label-free quantitation) and coupled (label-dependent quantitation) methods. Aligning the features of LC−MS maps directly for the decoupled method or by matching an LC−MS map and its virtual map for the coupled method results in a list of common features for replicate samples. We find that the ratio distribution of the common features successfully indicates the reproducibility of each experiment prior to MS/MS peptide sequencing in three different quantitation strategies: decoupled, coupled isotope-coded affinity tag, and coupled stable isotope labeling of amino acids in cell culture experiments.