Proteins are essential building blocks of living organisms and main components of nearly all metabolic pathways. The proteome of a cell is a highly dynamic entity and systematic protein profiling is important to study cellular regulations under regular and disease conditions.
The CECAD Proteomics Core Facility applies state-of-the-art methods of mass spectrometry to analyze the aging process on the protein level. Users submit projects in our online portal and deliver prepared samples to the facility. The samples are analyzed based on specific needs of the customers as determined in preliminary discussion following the facility guidelines. After measurement, proteins intensities are determined by powerful software tools and the facility performs basic statistical data evaluation as well as post-analysis consulting. All data will be stored by the RRZK following GLP principles. Further support is offered to customers during manuscript submission.
The proteomics platform uses the shotgun proteomics approach. Here, sample processing starts with cell lysis, fractionation, and digestion of the proteins. The resulting peptide mixture is separated by reversed phase chromatography using nano-UHPLC system. As the peptides elute from the column they are ionized and detected by a quadrupole Orbitrap hybrid mass spectrometer. Depending on specific questions, the facility offers various discovery or targeted approaches for data acquisition. In data-dependent acquisition (DDA) mode, masses of intact peptides are detected by MS scans which are followed by peptide fragmentation to determine the sequence and post-translational modifications of the peptides. In data-independent acquisition, the whole mass range is scanned in broad windows and all peptides present are fragmented at once. Fragments are later linked back to peptides with the help of a pre-build spectral library, making DIA the ideal tool for large studies with (relatively) similar samples. In contrast, parallel reaction monitoring (PRM) enhances the specificity of the measurements for a small number of pre-chosen peptides, making it the ideal tool for targeted analyses.
