MSE quantified stroma proteome

Label-free protein quantification has developed into an attractive alternative to isotopic labeling for the quantification of proteins by mass spectrometry. Recently, the suite of label-free quantification strategies was expanded by LC-MSE–based absolute and relative protein quantification. We report here a systematic evaluation of high definition (HD) MSE-based protein quantification and identification with the chloroplast stroma proteome. This proteome is of high complexity and comprises a wide dynamic range of protein concentrations. Our analysis identified many chloroplast proteins that were not previously identified in large-scale proteome analyses, suggesting HD-MSE as a suitable complementary tool for discovery proteomics. We find that HD-MSE tends to underestimate protein abundances at concentrations above 30 fmol, which is likely due to ion transmission loss and detector saturation. This limitation can be circumvented by combining HD-MSE and standard MSE scan types. The selection of peptides for protein quantification depends on sample characteristics; therefore different peptides may be used for the quantification of one protein in different replicates. This influences the robustness of protein quantification and requires critical scrutiny of quantification results. Based on the quantification of chloroplast stroma proteins we performed a meta-analysis and compared published quantitative data with our results, using a parts per million normalization scheme. Important pathways in the chloroplast stroma show quantitative stability against different experimental conditions and different quantification strategies.