Giovanni Cuda, MD

Clinical Proteomics

 

Proteomics is a relatively young discipline whose main purpose is to qualitatively and quantitatively characterize the protein content of a cell, tissue or body fluid. Furthermore, it promises to comprehensively unravel how proteins interact in macromolecular complexes, and how they are post-translationally modified. Compared to the first “omics” global effort, the sequencing of the human genome, proteomics poses different and greater challenges, because proteins are not amplifiable in the laboratory, like DNA. Besides, compared to nucleic acids, the primary structure of proteins is much more complex, and their range of concentrations in a biological sample is extremely wide. Nevertheless, because the main players in the chemistry of life are actually proteins, meeting those challenges promises to be very rewarding: proteomics has just began a journey during which many major contributions to the Biomedical sciences are expected to occur.

 

The large-scale study of proteins in a biological sample can be essentially accomplished by two complementary techniques. The first, in order of appearance, is two-dimensional gel electrophoresis (2DE), which resolves intact proteins by their isoelectric point and, in the second dimension, by their mass. Proteins are then visualized by a staining method, ultimately providing a quantitative two-dimensional “snapshot” of a selected proteome. The second approach, gel-free, is the so-called “bottom-up” or “shotgun” proteomics. Many variants of this approach can be adopted, but, in general, the proteome under investigation is analysed by performing enzymatic digestion of the whole protein mixture and subsequently performing, on the resulting peptides, multidimensional chromatographic separations interfaced to tandem mass spectrometry (MS/MS). Proteins are chopped into peptides because this procedure allows for better chromatographic separations of the analytes and for more efficient MS/MS analysis, capable to fully determine the amino acid sequence of polypeptides shorter than 40-50 amino acids. Though the bottom-up approach is not able to provide information about intact protein isoforms, obtainable by 2DE, this second approach is faster, more comprehensive in terms of detected gene products, and more prone to automation.

 

The Laboratory of Proteomics and Mass Spectrometry at the University “Magna Graecia” of Catanzaro was established in 2004.

It is currently equipped with state-of-the art proteomics technologies for performing proteomic analysis both via 2DE and via the “shotgun” approach. Instrumentation comprises a differential in gel electrophoresis system (DIGE) for 2DE analysis and two Q-Exactive mass spectrometers interfaced with nanoscale liquid chromatography (nanoLC-Q-ToF) for “shotgun” proteomics and for protein identification. Over the past years, the  Laboratory of Proteomics and Mass Spectrometry has gained extensive knowledge in applications such as proteomic analysis of biological fluids, mainly human plasma/serum, characterization of post-translational modifications (PTMs) on gel-separated proteins, and protein-protein interactions studies. Besides conducting independent research projects, the Laboratory has provided key contributions to several collaborative projects established between the laboratory itself and academic research groups, both internal and external to University of Magna Graecia.

While having well-served basic biomedical research over the past years, the actual long-term objective of the laboratory of Proteomics and Mass Spectrometry is to fully integrate proteomics technologies in clinical research, and, possibly, in the clinical analysis routine for specific tasks. In order to advance towards this goal, we are being upgrading our technology for achieving better sensitivity and throughput.

 

Proteolab@UMG is open for establishing new collaborations or providing service work for academic or industrial partners. We can offer:

 

• Identification of proteins separated by gel electrophoresis;

• Characterization of protein complexes by nanoLC-MS/MS via the shotgun approach;

• Proteomic analysis of biological fluids by 2DE.