Frequently asked questions
What is proteomics?
What is a biomarker?
The term “proteome” was coined by Marc Wilkins in 1994 and describes the entire protein complement of the genome produced by an organism. Proteomics, therefore, is the study of the structure and function of proteins and their associated post-translational modifications. This mixture of technical disciplines is becoming increasingly important as most disease states manifest at the protein level. Discovering and tracking aberrant changes in proteins can lead to drug therapies and other medicinal benefits in many different diseases.
What is shotgun proteomics?
The official National Institute for Health (US) definition is “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacologic response to a therapeutic intervention”.
How do I avoid keratin contamination?
Shotgun proteomics is the term given to the technique of analysing a complex mixture using chromatographic separation followed by mass spectrometry. The term is analogous to Craig Venter’s shotgun approach to sequencing the human genome, in that shotgun proteomics involves breaking proteins into constituent peptides, which are identified by mass spectrometry, and the peptide-level information is re-assembled into protein-level information. This technique is routinely used in the Facility for plasma, serum, CSF and other complex samples.
How do I avoid PEG contamination?
Keratin is a protein that is literally everywhere. It is present in skin, hair, dust and in clothing e.g. wool. Dust that settles around your preparation area and on your equipment will in time be contaminated by keratin. This can get into the sample preparation and will be detected by the mass spectrometer, in certain cases, it can influence the result of the sample when analysed by LC/MS/MS.
To reduce keratin contamination, make sure all sample areas are clean and any equipment used is rinsed and cleaned with high purity water. Ensure all solutions are fresh and stoppered to prevent dust or fibres from getting in to the bottles. If spots or bands are to be cut from gels, avoid touching the gel with bare hands. Avoid leaning over samples or gels and use fresh gloves that have been rinsed in high purity water.
Polyethylene glycol (PEG) can be a problem for LC/MS/MS which can lead to ion suppression of the peptide signals and interfere with protein/peptide identification. PEG contamination can occur from plastics used during sample preparations that involve high amounts of solvents such as acetone and methanol. Leeching of PEG from sample tubes etc may not normally interfere with results after sample processing for techniques such as immunoprecipitation or ELISA, however, PEG binds strongly to the C18 reversed-phase packing in the columns used to separate the peptides before entry into the mass spectrometer. The increasing solvent amounts during the LC gradients cause a prolonged release of the PEG from the column.
To avoid PEG contamination, try using plastics that can handle high solvent concentrations or use glass wherever possible. Plastic vials can be washed twice with methanol before use if contamination may be suspected, to remove possible residue from vial manufacture.