Conifer Protein Database
In collaboration with the UVic/Genome BC Proteomics Centre, we have developed a conifer protein database for proteomics consisting of translated sequences corresponding to all contigs and singletons derived from the complete spruce EST collection (updated March 1, 2007) and spruce FLcDNAs. Several formats of this database have been explored for compatibility with various protein identification platforms, including Mascot (Matrix Science Inc., Boston, MA), The Global Proteome Machine (Global Proteome Machine Organization), and ProteinPilot (Applied Biosystems, Foster City, CA). A corresponding reverse database has been derived for estimation of false positive protein identification rates. This database will be made public upon completion in agreement with our data release policy (Please contact Joerg Bohlmann for inquiries).
LC-MS/MS Data Analysis
We have established bioinformatics methods for the analysis of data derived from multidimensional LC-MS/MS experiments (i.e., gel-free methods). The Trans Proteomic Pipeline (The Institute for Systems Biology, Seattle, WA) has been adapted to work with Mascot interpreted data for the extraction and inspection of quantitative values for large-scale proteomics datasets. Standard operating procedures have been established for the routine analysis of iTRAQ (isotopic Tags for Relative and Absolute Quantitation), and SILAC (Stable Isotope Labeling with Amino acids in Cell culture) data. This required the development of novel perl scripts for successful implementation and was accomplished in collaboration with the Institute for Systems Biology and the UVIC/Genome BC Proteomics Centre.
Proteome Analysis of Weevil-Induced Sitka Spruce
We have performed a proteome analysis of weevil-induced Sitka spruce bark tissues with a comparison of untreated, mechanically wounded, and insect treated tissues at three time points (2, 6 and 48 hours). Over 1,200 proteins were quantitatively analysed, and 104 were found to be differentially expressed. 72% of differentially expressed proteins were successfully identified by LC-MS/MS analysis in combination with Mascot based database searching (Lippert et al., 2007). This same material was used in microarray studies by our project, enabling integrated protein and expression data.
Chitosan Elicitation in Spruce
We have performed an investigation of the effects of chitosan elicitation on spruce suspension cell cultures in collaboration with Max Planck Institute (see above for gene expression profiling). Samples represented eight biological replicates at five time points. A quantitative proteome analysis was performed using iTRAQ reagents and multiple reactions were performed for complete, balanced analysis of the samples. Each reaction consisted of 45 LC-MS/MS runs, and incorporated a novel iterative analysis methodology that facilitated deeper investigation of the spruce proteome. This method resulted in improved overlap between reactions and a corresponding improvement in the replication of a shotgun proteomics dataset. A total of 3,612 distinct proteins were identified with at least one high scoring peptide, while 1,863 were identified and quantitatively measured at least three times for every time point.
Proteotypic Peptides and Quantitative Analysis of Terpenoid Synthases
We have developed a panel of proteotypic peptides and methods for the targeted, rapid, quantitative analysis of terpenoid synthases (TPS) in complex samples. LC-MS/MS analysis of recombinant expressed and purified TPS was used to empirically determine and predict proteotypic peptides from Norway spruce and Sitka Spruce enzymes. Each proteotypic peptide provides a mass signature that is specific to a single TPS and allows the measurement of that enzyme in a complex protein sample with sub-picomole sensitivity and minimal sample handling. This method has been successfully applied to Norway spruce cell culture samples, in which distinct TPS could be detected and quantified. These proteins had not previously been detected in these samples using other methods. The analysis requires 90 minutes per sample and can now be scaled to include larger numbers of target proteins and can be applied to larger field trials. For further information, please contact Joerg Bohlmann.