title: Plant proteogenomics: from protein extraction to improved gene predictions.
abstract: Historically many genome annotation strategies have lacked experimental evidence at the protein level, which and have instead relied heavily on ab initio gene prediction tools, which consequently resulted in many incorrectly annotated genomic sequences. Proteogenomics aims to address these issues using mass spectrometry (MS)-based proteomics, genomic mapping, and providing statistical significance measures such as false discovery rates (FDRs) to validate the mapped peptides. Presented here is a tool capable of meeting this goal, the UCSD proteogenomic pipeline, which maps peptide-spectrum matches (PSMs) to the genome using the Inspect MS/MS database search tool and assigns a statistical significance to the match using a target-decoy search approach to assign estimated FDRs. This pipeline also provides the option of using a more reliable approach to proteogenomics by determining the precise false-positive rates (FPRs) and p-values of each PSM by calculating their spectral probabilities and rescoring each PSM accordingly. In addition to the protein prediction challenges in the rapidly growing number of sequenced plant genomes, it is difficult to extract high-quality protein samples from many plant species. For that reason, this chapter contains methods for protein extraction and trypsin digestion that reliably produce samples suitable for proteogenomic analysis.
authors.affiliation: Centre for Comparative Genomics, Murdoch University, Perth, WA, Australia.
authors.forename: Brett
authors.initials: B
authors.lastname: Chapman
authors.forename: Natalie
authors.initials: N
authors.lastname: Castellana
authors.forename: Alex
authors.initials: A
authors.lastname: Apffel
authors.forename: Ryan
authors.initials: R
authors.lastname: Ghan
authors.forename: Grant R
authors.initials: GR
authors.lastname: Cramer
authors.forename: Matthew
authors.initials: M
authors.lastname: Bellgard
authors.forename: Paul A
authors.initials: PA
authors.lastname: Haynes
authors.forename: Steven C
authors.initials: SC
authors.lastname: Van Sluyter
mesh_terms: D000465:Algorithms; D002853:Chromatography, Liquid; D002874:Chromosome Mapping; D030562:Databases, Protein; D018745:Genome, Plant; D023281:Genomics; D013058:Mass Spectrometry; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis* / Q000235:genetics*; D010944:Plants / Q000737:chemistry* / Q000235:genetics*; D040901:Proteomics; D057386:Search Engine
chemical_list: D010455:Peptides; D010940:Plant Proteins