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We have established a proteome reference map for Medicago truncatula root proteins using two‐dimensional gel electrophoresis combined with peptide mass fingerprinting to aid the dissection of nodulation and root developmental pathways by proteome analysis. M. truncatula has been chosen as a model legume for the study of nodulation‐related genes and proteins. Over 2500 root proteins could be displayed reproducibly across an isoelectric focussing range of 4–7. We analysed 485 proteins by peptide mass fingerprinting, and 179 of those were identified by matching against the current M. truncatula expressed sequence tag (EST) database containing DNA sequences of approximately 105 000 ESTs. Matching the EST sequences to available plant DNA sequences by BLAST searches enabled us to predict protein function. The use of the EST database for peptide identification is discussed. The majority of …