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Mass Spectrometry Unit


Staff: , , Carmine Cirillo

Mass Spectrometry (MS) based proteomics analysis unravels what proteins do: the signaling pathways in which they are involved, the other proteins with which they interact and the post-translational modifications they are subjected to. In general, a cell phenotype reflects the proteome status, so the mass spectrometry studies can provide fundamental biological insights on molecular mechanims.

At TIGEM, we are starting a proteomics core allowing our researchers to quantitatively describe signaling networks and determine how components of signaling cascades are affected in response to pathway perturbations. We perform complementary qualitative and quantitative MS approaches for identification and quantitation of proteins and their post-translational modifications (PTMs), both at small (e.g. interactomes) and large (whole cell- or subcellular proteome) scale.
In general, we follow a standard approach: protein samples are first treated with proteases (e.g. Trypsin and/or LysC) and then analyzed by LC-MS/MS for identification and quantification of the resulting peptides and thus the underlying proteins. Besides label-free protein quantitation based on summed peak intensities of identified peptides, we take advantage of various labelling techniques, like Stable Isotope Labelling by/with Amino acids in Cell culture (SILAC) and Tandem Mass Tag (TMT) reagents. The generated spectral data is mostly analyzed using Max Quant (MPI Martinsried) and Proteome Discoverer (Thermo Scientific) in order to obtain the list of identified proteins and their abundances in the samples.

Our facility is equipped with a Q Exactive HF Hybrid Quadrupole-Orbitrap with an ultra-high-field analyzer. It provides higher sensitivity and acquisition speed for highly complex samples, especially in conjunction with the hyphenated UHPLC which allows the use of smaller column particles and longer columns due to higher pressure resistance of the fluidics.