Phylogeny of Pristionchus pacificus and proteogenomics workflow employed in this study. (A) Phylogenetic relationship of nematodes with sequenced genomes. The genome sizes (megabases) are written in brackets. The clades are depicted in the tree. (B) P. pacificus gene expression was assessed at the levels of transcription and translation and in three different developmental stages: dauer, J2, and “mixed stage” (containing all developmental stages, including eggs). In proteomics approach, several workflows for protein extraction and separation were used. ESTs detected with 454 pyrosequencing and peptide sequences detected with LTQ-Orbitrap mass spectrometry were used for genome reannotation.