Non Gel Based Workflows (Shotgun Proteomics)
There are many occasions when gel based techniques are inappropriate for proteomic studies such as when analyzing membrane proteins or when samples are complex and require pre-fractionation or when quantification or proteome comparison is measured at the peptide level using MS or MS-MS data on labeled or unlabelled peptides. We have extensive experience in these shotgun approaches using the instrumentation available in the proteomics facility at Durham. (publications).
Typically protein extracts are prepared from cells, tissue or organelle preparations and these are digested directly with trypsin in solution to give a complex peptide digest. This can then be labeled with ITRAQ (Applied Biosystems) reagents if relative quantification is required or analyzed without labeling if only protein identification is required. A strong cation exchange fractionation of the peptide digest is then performed on a GE Healthcare Ettan micro LC chromatography system resulting in up to 40 simplified peptide fractions for LC-MS-MS analyses either on the QSTAR or the 4800 TOF-TOF instrument. Peptide fractions for LC-MS-MS analyses are further separated on a nano reverse phase column on a GE Healthcare MDLC system coupled to the nanospray source of the QSTAR for nano-ESI tandem mass spectrometer analyses or nano-LC reverse phase separated and spotted onto MALDI target plates using an Applied Biosystems LC-TEMPO for LC-MALDI-TOF-TOF analyses. Data is acquired throughout the peptide elution from the columns and data acquisition and spectral processing is carried out using Applied Biosystems Analyst and BioAnalystTM or GPS explorer software respectively.
Protein identification is achieved using MASCOT search tools on in house computers searching all publicly available databases and comprehensive data processing and quantification of whole shotgun experiments is carried out using Applied Biosystems Protein Pilot software.