The identification of proteins contained in the biological sample is done through LC-MS/MS analysis (liquid chromatography coupled to tandem mass spectrometry). For this, peptides resulting from trypsin digestion are injected onto a reverse-phase liquid chromatography column (C18), which retains peptides based on hydrophobicity. A gradient of acetonitrile is then applied to gradually elute the peptides throughout the analysis duration (5 to 120 minutes).
The output of the column is directly connected to the electrospray source of the mass spectrometer, where peptides are ionized. The mass-to-charge ratio (m/z) of the ions thus obtained is then measured by the mass analyzers of the instrument to obtain a so-called “MS1 spectrum.” It is also possible to fragment the ionized peptides in the collision cell of the instrument to obtain fragmentation spectra called “MS2 spectra.” The mass spectrometer acquires thousands of MS1 spectra and tens of thousands of MS2 spectra during the analysis duration.
All the MS1 and MS2 spectra obtained at the end of the analysis are then processed by software (e.g., Mascot) to compare the experimental masses they contain to theoretical masses calculated from publicly available sequence databases. The software also groups identified peptides to obtain a list of proteins and validates the results at a 1% False Discovery Rate (less than 1% false positives).
The results are provided in Scaffold format, which allows visualization of the list of identified proteins and corresponding peptides. It also contains spectral counting values, which can provide an approximate idea of the protein’s abundance in a sample compared to another.