A label-free differential quantitative mass spectrometry method for the characterization and identification of protein changes during citrus fruit development

Protein changes in citrus juice sac cells were identified and quantified using label-free shotgun methodologies. Two alternative methods, differential mass-spectrometry (dMS) and spectral counting (SC) were used to analyze protein changes occurring during earlier and late stages of fruit development. Both methods were compared in order to develop a proteomics workflow that could be used in a non-model plant lacking a sequenced genome. In order to resolve the bioinformatics limitations of EST databases from species that lack a full sequenced genome, we established iCitrus. iCitrus is a comprehensive sequence database created by merging three major sources of sequences (HarvEST:citrus, NCBI/citrus/unigenes, NCBI/citrus/proteins) and improving the annotation of existing unigenes. iCitrus provided a useful bioinformatics tool for the high-throughput identification of citrus proteins. We have identified approximately 1500 citrus proteins expressed in fruit juice sac cells and quantified the changes of their expression during fruit development. Our results showed that both dMS and SC provided significant information on protein changes, with dMS providing a higher accuracy.Our data supports the notion of the complementary use of dMS and SC for label-free comparative proteomics, broadening the identification spectrum and strengthening the identification of trends in protein expression changes during the particular processes being compared.Fruit ripening and development has being studied using transcriptomic, proteomics, and metabolomics approaches [1-8]. Quantitative proteomics provides an alternative approach for studies of fruit development. In the last few years, quantitative proteomics has been widely applied for the quantification of complex biological samples [9-11]. The most commonly used approach for comparative proteomic analysis of plant tissues is the application of 2DE-gels. This method is limited in sensitivity, has a low dynamic range, it is inefficient when a