AbstractVarious functional genomics platforms are required to define the phenotype associated with a mutant. Global protein analyses may be included in any study. We describe here a rapid method of protein sample preparation and analysis, suitable for all laboratories and using Arabidopsis plantlets as the starting material. This reliable and reproducible method for high yield protein extraction from small amounts of material can be used on even the most recalcitrant tissues. The proteins extracted are suitable for many types of protein analysis, including nondenaturing investigations. This method was validated by a rigorous 2‐DE approach, coupled with unambiguous LC‐MS/MS identifications featuring strong sequence coverage (average of 26% with eight different peptides/spot protein). The reproducibility of the method was demonstrated by multiple protein identifications from identical series of spots. An interactive map (http://www.isv.cnrsgif.fr/gel2d/), including 435 protein variants showed that (i) 38% of the proteins were yet unreported, (ii) reduced subfractionation, (iii) had frequent protein modifications (average of two spots/protein entry), and (iv) underwent no major proteolytic events other than leader peptide cleavage. Finally, a simple mobility shift method for the large subunit of RuBisCo (LS) in the first dimension made it possible to characterize previously masked protein spots.