Expressed Peptide Tags:  An Additional Layer of Data for Genome Annotation

Abstract

While genome sequencing is becoming ever more routine, genome annotation remains a challenging process. Identification of the coding sequences within the genomic milieu presents a tremendous challenge, especially for eukaryotes with their complex gene architectures. Here, we present a method to assist the annotation process through the use of proteomic data and bioinformatics. Mass spectra of digested protein preparations of the organism of interest were acquired and searched against a protein database created by a six-frame translation of the genome. The identified peptides were mapped back to the genome, compared to the current annotation, and then categorized as supporting or extending the current genome annotation. We named the classified peptides Expressed Peptide Tags (EPTs). The well-annotated bacterium Rhodopseudomonas palustris was used as a control for the method and showed a high degree of correlation between EPT mapping and the current annotation, with 86% of the EPTs confirming existing gene calls and less than 1% of the EPTs expanding on the current annotation. The eukaryotic plant pathogens Phytophthora ramorum and Phytophthora sojae, whose genomes have been recently sequenced and are much less well-annotated, were also subjected to this method. A series of algorithmic steps were taken to increase the confidence of EPT identification for these organisms, including generation of smaller subdatabases to be searched against, and definition of EPT criteria that accommodates the more complex eukaryotic gene architecture. As expected, the analysis of the Phytophthora species showed less correlation between EPT mapping and their current annotation. While approximately 76% of Phytophthora EPTs supported the current annotation, a portion of them (7.7% and 12.9% for P. ramorum and P. sojae, respectively) suggested modification to current gene calls or identified novel genes that were missed by the current genome annotation of these organisms.

Profiling the Secretome and Extracellular Proteome of the Potato Late Blight Pathogen Phytophthora infestans

“…Thirteen proteins with a life stage specific expression pattern were identified by 2D-gel electrophoresis, including CRN2 (31). For P. sojae and P. ramorum, a global proteomic approach was used to detect proteomic differences between life stages (32,33), and a recent large-scale phosphoproteome analysis revealed the phosphorylation status of thousands of proteins and provided novel information on life stage specific phosphorylation events in P. infestans (34). Despite their importance, proteomic studies on Phytophthora extracellular proteins are even more limited.…”

Discovery and revision of Arabidopsis genes by proteogenomics

“…Proteogenomics (using proteomic information to annotate the genome) complements nucleotide-based annotation in that it unambiguously determines reading frame, translation start and stop sites, splice boundaries, and the validity of short ORFs. By combining DNA-based annotation with proteogenomics, an accurate and more complete protein-coding catalog can be obtained (6)(7)(8)(9)(10). With its clear potential for improving genome annotation, proteogenomics could be integrated with genome projects.…”

Comparative proteogenomics: Combining mass spectrometry and comparative genomics to analyze multiple genomes

“…However, while there are hundreds of studies on using ESTs for genome annotation, EPT studies are still in infancy (Savidor et al 2006). This is unfortunate since EPTs may provide some advantages over ESTs and are easy to generate.…”