Center for Clinical proteomics has access to a large variety of mass spectrometers and technologies in that relation of which many are state-of-the-art.
Electrospray (ESI) MS instruments:
The CCP has access to LTQ-Orbitrap XL and Velos Mass Spectrometers (Thermo Scientific) for high resolution and high accuracy tandem mass spectrometry of peptides and proteins. The instruments are equipped with the capability to perform advanced peptide/protein fragmentation using Collision Induced Dissociation (CID), Pulsed-Q Collision Induced Dissociation (PQD), higher-energy C-trap dissociation (HCD) and electron transfer dissociation (ETD)
The CCP has in addition access to the QTOF Premier (Waters), QTOF Synapt (Waters) and QTOF micro (Waters) for high resolution and high accuracy tandem mass spectrometry of peptides and proteins. The instruments are capable of performing Collision Induced Dissociation (CID) and MSE identification and quantitation of peptides.
TSQ Vantage Mass Spectrometers (Thermo Scientific) are available for Selected Reaction Monitoring (SRM/MRM) of peptides.
Matrix assisted laser desorption/ionization (MALDI) MS instruments:
CCP has access to a number of MALDI MS instruments:
- Ultraflex MALDI TOF-TOF Mass spectrometer (Bruker Daltonics)
- 4800 TOF-TOF Mass Spectrometer (ABSciex)
- MALDI STR workstation (ABSciex)
- MALDI QTOF Mass Spectrometer (Waters)
In recent years large scale mass spectrometry driven proteomics has become increasingly popular and new powerful instrumentation has been developed, increasing sensitivity, data quality and protein identification throughput. From being an exclusively qualitative technique, mass spectrometry is now becoming a widely accepted quantitative technique in proteomics.
In CCP we have a wide range of techniques available for performing quantitative proteomics. These include:
- Chemical labelling of peptides or proteins using iTRAQ (comparison of up to 8 different samples), TMT tag (comparison of up to 6 different samples) or dimethyl labelling (comparison of up to 3 different samples). These technologies are directly applicable to all kind of samples including primary cell material, body fluids and biopsies.
- Metabolic labelling of cell cultures (e.g., SILAC). These strategies are not directly applicable to the analysis of primary biological material as the stable isotope amino acids are incorporated during cell growth. Heavy labelled animals have been reported for quantitative proteomics.
- Label free proteomics. Here no labels are used and the quantitation is relying on for example extracted ion chromatograms.
Many proteins are post-translationally modified and it is those modifications that often determine the function, interaction and physio-chemical properties of these proteins. A posttranslational modification (PTM) is the covalent attachment of a chemical group to a protein after protein synthesis or the proteolytical removal of a signal peptide after localization. PTMs are essential for the function and localization of proteins. Therefore, the analysis of PTMs in clinical proteomics might provide new discoveries both in the disease understanding and in the biomarker discovery areas.
CCP has a world recognized experience in identification, characterization and quantitation of various PTMs, including phosphorylation, glycosylation, acetylation, etc. We have robust protocols for quantitative analysis of the above mentioned PTMs and are routinely identifying thousands of phosphorylation sites and sialylated glycosylation sites from low amount of primary cell material.