discovery and annotation of small proteins using genomics proteomics and computational approaches small proteins 10200 amino acids aa in length encoded by short open reading frames sorf play important regulatory roles in various biological processes including tumor progression stress response flowering and hormone signaling however ab initio discovery of small proteins has been relatively overlooked recent advances in deep transcriptome sequencing make it possible to efficiently identify sorfs at the genome level in this study we obtained 26 million expressed sequence tag est reads from populus deltoides leaf transcriptome and reconstructed fulllength transcripts from the est sequences we identified an initial set of 12852 sorfs encoding proteins of 10200 aa in length three computational approaches were then used to enrich for bona fide proteincoding sorfs from the initial sorf set 1 codingpotential prediction 2 evolutionary conservation between p deltoides and other plant species and 3 gene family clustering within p deltoides as a result a highconfidence sorf candidate set containing 1469 genes was obtained analysis of the protein domains nonproteincoding rna motifs sequence length distribution and protein mass spectrometry data supported this highconfidence sorf set in the highconfidence sorf candidate set known protein domains were identified in 1282 genes higherconfidence sorf candidate set out of which 611 genes designated as highestconfidence candidate sorf set were supported by proteomics data of the 611 highestconfidence candidate sorf genes 56 were new to the current populus genome annotation this study not only demonstrates that there are potential sorf candidates to be annotated in sequenced genomes but also presents an efficient strategy for discovery of sorfs in species with no genome annotation yet available