The Laboratory offers a possibility to perform a global quantitative comparison of two or more proteomes with Orbitrap Velos mass spectrometers. Depending on experimental design and acceptable costs, various high-throughput analytical strategies can be used, including:
- label-free method - based on quantitative LC-MS measurement of samples and subsequent comparison of LC peak heights of peptide signals. This type of analysis is less expensive (no additional sample preparation) and suitable for long series of samples. It is, however, not compatible with additional offline peptide separation techniques (such as isoelectrofocusing), therefore restricted to less complex peptide/protein mixtures. Protein concentration in samples for label-free analysis should be at least 1 µg/µl and the total protein amount should be no less than 3 µg.
- SILAC - this isotope labeling-based method allows for a common LC-MS measurement of two samples. Experimental cell cultures are labeled using culture media which contain stable isotopes (13C or 15N) icorporated into certain amino acids (i.e. lysin or arginin). SILAC analysis requires appropriate changes in experiment design and is resticted to certain sample types.
- iTRAQ - a labeling-based method which allows for simultaneuos measurement of 4 or 8 samples. In this approach, stable isotope labeling is performed along with routine trypsin digestion, therefore no adjustments in experiment design are required. Highly complex samples can be previously fractionated using offline techniques like isoelectrofocusing, provided enough sample amount (c.a. 100 µg) is available. iTRAQ analysis is suitable for complex protein mixtures, like whole cell organells or tissues.
Differential proteomics experiments performed in the Laboratory are accompanied by extensive validation and statistical analysis, performed by the staff.
For information on costs and terms of global analysis of proteomes, please contact the Head of the Laboratory