Gene Expression (The process by which a gene's coded information is converted into the structures present and operating in the cell. Expressed genes include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but not translated into protein (e.g., transfer and ribosomal RNAs)).
Genotyping (Testing that reveals the specific alleles inherited by an individual; particularly useful for situations in which more than one genotypic combination can produce the same clinical presentation, as in the ABO blood group, where both the AO and AA genotypes yield type A blood ).
- SNP Genotyping
- Affymetrix GeneChip Technology
- Allelic Discrimination Using the 5' Nuclease Assay
Genome Level Analysis
- CHIP- Qauantitative PCR-qCHIP
- CHIP on Chip
- Array CGH
The Proteomics Laboratory of the Department of Medicine is an intricate part of Life Sciences Technology Laboratory within the Personalized Medicine Research Program. Its’ aim is to provide users with protein separation and identification services. The center is based around two proteomics systems: a Thermo Electron Finnigan LTQ mass spectrometer and a Beckman Coulter Proteome Lab PF2D chromatography system both of which are described further below.
Thermo Electron Finnigan LTQ mass spectrometer
The LTQ is a very sensitive linear ion trap mass spectrometer. In our facility the LTQ is integrated with a Thermo Electron Surveyor quaternary HPLC system and a Spark Holland Micro Autosampler. Current ionization methods available for the LTQ are electrospray ionization (ESI) and nano electrospray ionization (NSI). We perform peptide mass fingerprinting (PMF) using NSI which is the standard approach to identifying proteins on this type of instrument. Briefly the protein or protein mixture is digested with trypsin and the tryptic peptides separated by revered phase (RP) resin packed into a fused silica capillary directly into the LTQ using gradients designed to accommodate the complexity of the sample. Peptides are identified by MSn (where n= 2-10 depending on the initial complexity of the sample) using the data-dependent data acquisition function of the LTQ. The amino acid sequence of the tryptic peptides can be determined from the MSn data by an algorithm that is part of the Sequest software containe in the Bioworks suite (for which our group has a license and a dedicated computer for data analysis).
Beckman Coulter Proteome Lab PF2D chromatography system.
It consists of two high precision HPLC systems and two fraction collectors. The system is set up to fractionate highly complex samples such as tissue, cell or plasma lysates by chromatofocusing which is used as the first dimension of fractionation. Fractions collected from the first dimension are injected automatically onto the second dimension column which in this case is RP resin and fractions are collected by the second fraction collector. The PF2D has very sophisticated software that allows second dimension chromatograms from two different but related samples (such as treated and untreated populations of cells or wild type and knockout cells) to be compared up by overlaying one on the other. In this way differences in chromatographic peak profiles between the two samples can be easily detected. Peaks where there are significant differences can be retrieved from their collection plates and the proteins from each peak prepared for identification by peptide mass fingerprint analysis on the LTQ mass spectrometer.
Method of operation of the Proteomics Laboratory
- The Proteomics Center is headed by Avelino Teixeira Ph.D.
- The Center’s aim is to provide flexible and ongoing proteomics services to the DOM and its collaborators. The majority of the projects will be undertaken on a case by case basis as a collaborative effort.
- The costs of the initial work will be determined by the Proteomics Lab and if successful and used in a grant proposal, the expectation is that the Proteomics Lab will be included as a percent effort in that proposal for the collaboration to continue.
- To initiate collaboration, the PI should meet with Dr. Teixeira (X43110) to discuss the overall project and decide if and how the Proteomics Lab can assist in the project.
- Agreement to collaborate will be formalized in a brief proposal describing the objectives of the work and the methods to be applied by the Proteomics Lab.
- The PI should assign a member of their lab as contact person to do the necessary upstream sample preparation prior to delivery to the Proteomics Lab.
- All upstream preparation should be discussed beforehand to ensure that it is done in the manner best suited for the proposed work.
- On receipt of the samples they will be logged and an estimate of time for start of the runs will be made.
- Data will be collected and put into a format that can be viewed and analyzed by the collaborating lab.
- Periodic meetings with the PI and contact person will be held to discuss results and plan future work.