Brassica napus (cultivar Reston) seed proteins were analyzed at 2, 3, 4, 5, and 6 weeks after flowering in biological quadruplicate using two-dimensional gel electrophoresis. Developmental expression profiles for 794 protein spot groups were established and hierarchical cluster analysis revealed 12 different expression trends. Tryptic peptides from each spot group were analyzed in duplicate using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatographytandem mass spectrometry. The identity of 517 spot groups was determined, representing 289 nonredundant proteins. These proteins were classified into 14 functional categories based upon the Arabidopsis (Arabidopsis thaliana) genome classification scheme. Energy and metabolism related proteins were highly represented in developing seed, accounting for 24.3% and 16.8% of the total proteins, respectively. Analysis of subclasses within the metabolism group revealed coordinated expression during seed filling. The influence of prominently expressed seed storage proteins on relative quantification data is discussed and an in silico subtraction method is presented. The preponderance of energy and metabolic proteins detected in this study provides an in-depth proteomic view on carbon assimilation in B. napus seed. These data suggest that sugar mobilization from glucose to coenzyme A and its acyl derivative is a collaboration between the cytosol and plastids and that temporal control of enzymes and pathways extends beyond transcription. This study provides a systematic analysis of metabolic processes operating in developing B. napus seed from the perspective of protein expression. Data generated from this study have been deposited into a web database (http://oilseedproteomics.missouri.edu) that is accessible to the public domain.Brassica napus (also known as rape and oilseed rape) is the third largest oilseed crop in the world, providing approximately 13% of the world's supply of vegetable oil. B. napus seeds produce oil and protein as the main storage compounds out of the three principal storage reserves (proteins, oil [triacylglycerols], and carbohydrates [starch]) found in plant seeds (Norton and Harris, 1975;Murphy et al., 1989;King et al., 1997;Schwender and Ohlrogge, 2002). These compounds support early seedling growth, and in nature the relative proportions of these compounds vary dramatically among different plant seeds. Seeds of legume species such as soybean (Glycine max) and pea (Pisum sativum) produce seeds with 20% to as much as 40% protein and 20% oil, while B. napus produces seeds with approximately 40% oil and 15% protein (Gunstone et al., 1995).Biochemical and molecular studies are beginning to define the biosynthetic pathways responsible for accumulation of these storage components in B. napus

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