*2.2.4. Shotgun MS/MS*

Shotgun proteomics is a widely used technique in proteomics for identifying proteins [57], with great sensitivity, making it a great influence in the discovery of clinically actionable biomarkers [58]. To perform it, a complex mixture of proteins is separated by sequence-specific proteolysis, forming peptides that will then be separated in smaller fragments to be later analyzed by mass spectrometry [57]. Each peptide will have a mass associated to it, but since peptides may have the same a.a., but in a different arrangement, it is important to assess the sequence, which can be done by the ratios of its mass spectrum, that is, by MS/MS [57].

This method is often disregarded by researchers, due to its early problems in reproducibility and fallible nature [58, 59]. However, the development of bioinformatic tools has allowed a decrease in these problems, making it a viable option for proteomic research [57–59].
