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Application of proteomics is becoming increasingly important to understand the function of genes and their encoding proteins. This is due to not only the poor correlation between the transcript levels and protein accumulation, but also the critical roles of posttranslational modifications that increase the functional diversity of the proteins and influence almost all aspects of plant growth and its response to the environment. This chapter describes the gel-based quantitative and comparative proteomics that combine two-dimensional gel electrophoresis with mass spectrometry analysis to detect, quantify, and characterize proteins and their posttranslational modifications with specific focus on analyzing nodule samples. This method is also applicable for other tissue types.