Proteomics

Science programme: Genomic technologies

HortResearch’s proteomics team is identifying key proteins in proteomes of plant and animal tissues. A ‘proteome’ is the protein complement of the genome, providing a fingerprint for each cell or tissue type at a point in time and reflecting a defined physiological and developmental state. There are several thousand proteins in a typical proteome.

Our scientists use modern technology to identify specific candidates of interest that show developmental variation or changing properties. They then analyse these using a variety of specialised chromatographic and mass spectrometric methods.

Identification of these proteins is important as it assigns provisional function to the genes that encode them, and their presence, absence or modification is a consequence of the expression of these genes. Proteomics therefore provides an essential element to the functional analysis pipeline for genes of interest to us.

As well as providing protein identification capability on a routine basis, our proteomics facility means we must develop new technologies to enable improvements and refinements to sample throughput, limits of sensitivity, data quality and the integration of results into the genomics pipeline.

The team is currently involved in four major projects:

• Proteomics development
• Plant/Pathogen interactions
• Proteins involved in Olfaction
• Spray‑free agriculture.

Our recent successes include:

• Identification of several proteins potentially involved in plant pathogenesis
• Defining a transcription factor from Rhizobium
• Enumeration of the complement of coat proteins from a novel virus
• Identification of allergenic proteins
• Phosphorylation analysis of rat brain proteins.

Our approach is one of intensive interrogation of data from targeted proteins rather than blanket coverage of the entire proteome. Using differential methods such as reactive fluorimetric dyes to label different samples separated on 2‑dimensional gels, we can examine in detail by mass spectrometry individual proteins showing changes in spot intensity or a shift in position.

Proteins isolated as stained gel plugs are digested with trypsin and analysed with our electrospray LCMS, a Thermo Finnigan LTQ ion trap mass spectrometer. The results are interpreted using TurboSEQUEST, a software package which pattern matches experimental MSMS spectra with predicted spectra of sequences from protein and translated nucleotide databases, resulting in sequence assignments to the constituent peptides.

Where only rudimentary databases are available for peptide sequence comparisons, we use the software package DeNovoX to provide de novo sequencing of peptides. We were the first group in Australasia to purchase this package.