What is Proteases and the Identification of Proteins

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Protein analysis and identification through mass spectrometry first requires a breakdown of each protein into their composite peptides. Once the protein has been broken down, the peptides can be separated through the use of a reverse phase column and the peptides and peptide fragments can be measured using a mass spectrometer.

Proteases and the Identification of Proteins
Breaking down proteins for analysis is performed through proteases, of which the most commonly used is trypsin. Proteases are designed to be sequence specific and are tailor-made to produce the best protein digestion. Alternative proteases are used for specific sequencing, whereas more versatile all-around proteases may be used throughout the process of mass spectrometry. Ideally, proteases produced for the use of mass spectrometry are able to accommodate multiple strategies for digestion and have been tested for use within mass spectrometry devices. When it comes to proteases, both the digestion time and cleavage specificity are the most important factors. Digestion time may impact the reliability of the mass spectrometry results, while cleavage will impact the separated peptides.

Proteases are produced in-solution, in-gel, and isolated as standalone proteases, to be more useful and convenient within a laboratory setting. Pro-teases that are provided in-solution are generally preferred for smaller samples, whereas in-gel suspensions are designed for more complex operations that may require additional control. Either way, the quality of the proteases are still equally validated, and the results of the proteases should be consistent.