Generation of a predicted protein database from EST data and application to iTRAQ analyses in grape (Vitis vinifera cv. Cabernet Sauvignon) berries at ripening initiation

Abstract

Background: iTRAQ is a proteomics technique that uses isobaric tags for relative and absolute quantitation of tryptic peptides. In proteomics experiments, the detection and high confidence annotation of proteins and the significance of corresponding expression differences can depend on the quality and the species specificity of the tryptic peptide map database used for analysis of the data. For species for which finished genome sequence data are not available, identification of proteins relies on similarity to proteins from other species using comprehensive peptide map databases such as the MSDB.

Results: We were interested in characterizing ripening initiation ('veraison') in grape berries at the protein level in order to better define the molecular control of this important process for grape growers and wine makers. We developed a bioinformatic pipeline for processing EST data in order to produce a predicted tryptic peptide database specifically targeted to the wine grape cultivar, Vitis vinifera cv. Cabernet Sauvignon, and lacking truncated N- and C-terminal fragments. By searching iTRAQ MS/MS data generated from berry exocarp and mesocarp samples at ripening initiation, we determined that implementation of the custom database afforded a large improvement in high confidence peptide annotation in comparison to the MSDB. We used iTRAQ MS/MS in conjunction with custom peptide db searches to quantitatively characterize several important pathway components for berry ripening previously described at the transcriptional level and confirmed expression patterns for these at the protein level.

Conclusion: We determined that a predicted peptide database for MS/MS applications can be derived from EST data using advanced clustering and trimming approaches and successfully implemented for quantitative proteome profiling. Quantitative shotgun proteome profiling holds great promise for characterizing biological processes such as fruit ripening initiation and may be further improved by employing preparative techniques and/or analytical equipment that increase peptide detection sensitivity via a shotgun approach.

Figure 1

Schema of the workflow in construction of the predicted peptide database based upon Vitis spp. ESTs. Steps in EST selection (yellow), EST curation (orange), contig assembly (green), and translation, start methionine prediction, tryptic cleavage site prediction, and removal of predicted truncated N- and/or C-terminal peptides (blue) are shown. The in-house "GrapeGen" EST database was derived from the V. vinifera cv. Cabernet Sauvignon and Muscat Hamburg cDNAs [17]. Where a CS EST was duplicated between the Genbank and in-house databases, the Genbank EST was removed and the EST from the in-house database, containing the phred scores, was used for clustering. CS = Cabernet Sauvignon ESTs; MH = Muscat Hamburg ESTs; Vv = Vitis vinifera; Wild = Vitis spp. ESTs other than V. vinifera. A key to the codes used in 'Cluster ORF ID' and 'Protein Annotation' in Additional files 1 through 8 is presented in Additional file 10 and may be printed out as a quick reference when examining the iTRAQ results; tissue types (i.e. CS_) are described in Methods and Additional file 10.

Figure 2

K-means cluster analysis of expression data for exocarp proteins. Four partitions were used to classify proteins that were increasing strongly (top left panel), increasing gradually (top right panel), not changing significantly (bottom left panel), or decreasing (bottom right panel) along ripening initiation. Ripening initiation stages corresponding to the expression data are depicted by the photographs on the two lower x-axes. X-axes on the lower two panels also correspond identically to the top two panels. Y-axes in the left two panels correspond identically to the right two panels. Numbers of proteins are shown in the top left corner of each panel.

Figure 3

K-means cluster analysis of expression data for mesocarp proteins. Four partitions were used to classify proteins that were increasing strongly (top left panel), increasing gradually (top right panel), not changing significantly (bottom left panel), or decreasing (bottom right panel) along ripening initiation. Ripening initiation stages corresponding to the expression data are depicted by the photographs on the two lower x-axes. X-axes on the lower two panels also correspond identically to the top two panels. Y-axes in the left two panels correspond identically to the right two panels. Numbers of proteins in each cluster are shown in the top left corner of each panel.

Figure 4

Model showing transcript (squares) and protein (circles) expression trends annotated with functions in ABA, BR, and anthocyanin biosynthesis in the grape berry pericarp at ripening initiation. Transcript accumulation data are based on previous studies [26,27,35-37], whereas all protein accumulation data presented here are new findings for grape berries. Green indicates an increase in transcript or protein abundance during ripening initiation. Yellow indicates no significant change in protein accumulation. ABA-O = abscisic aldehyde oxidase; ANS = anthocyanin synthase; BR6OX1 = brassinosteroid-6-oxidase; BRI1 = receptor-like kinase, brassinosteroid insensitive 1; β-hyd = β-carotene hydroxylase; CYD = cyanidin; CYTb5 = cytochrome b5; DFR = dihydroflavonol reductase; DHK = dihydrokaempferol; DHM = dihydromyricetin; DHQ = dihydroquercitin; DMADP = dimethylallyl diphosphate; DPD = delphinidin; DWF1 = dwarf 1; F-OMT = putative anthocyanin flavonoid O-methyltransferase; FDPS = farnesyl diphosphate synthase; F3H = flavonoid-3-hydroxylase; F3'H = flavonoid-3'-hydroxylase; F3'5'H = flavonoid-3'5'-hydroxylase; GCR2 = G protein-coupled receptor 2; GGDP = geranylgeranyl diphosphate; GGDPS = geranylgeranyl diphosphate synthase; IDP = isopentenyl diphosphate; IPI = isopentenyl diphosphate isomerase; LCY-β = lycopene-β-cyclase; MVD = malvidin; NCED = 9-cis-epoxycarotenoid dioxygenase; PDS = phytoene desaturase; PND = peonidin; PSY = phytoene synthase; PTD = petunidin; SDR = short-chain alcohol dehydrogenase/reductase; VDE = violaxanthin de-epoxidase; ZDS = ζ-carotene desaturase; ZEP = zeaxanthin epoxidase.