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Digestion of proteins or proteinaceous entities is a tedious process that is not reproducible and tainted with auto digests of enzymes used in the process.
Immobilized enzymes however have made it possible to avoid this and when immobilized on stable polymeric media they have paved the way to automation.
The present application note describes the details of the automated digestion of Cytochrome c from equine heart using a basic HPLC instrument
See Application Note 132:
There are however a number of variables that may affect the automated digestion.
The next application note considers such variables.
See Application Note 133:
Although the speed of digestion is high considering the linear velocities in a narrow bore column of 2.1 mm ID, the question is how fast, within practical flow rates, is the enzyme able to digest the substrate?
This is the subject of the following Application Note.
See Application Note 134:
Automated Digestion with StyrosZyme® TPCK-Trypsin, Immobilized Enzyme on Polymeric Hard Gel Simulated-Monolith™. Effect of Linear Velocity and Column Length on the Digestion of Oxidized Insulin B Chain.
The stability of the StyrosZyme® TPCK-Trypsin is tested for reproducibility as the goal of the automation is to run the digestion and mapping continuously and under control.
See Application Note 135:
Using the Agilent 1290 Infinity as a UHPLC the automated digestion can be run on Narrow Bore columns extending the number of runs with limited amount of solvent as well as minimum amount of sample.
See Application Note 136:
In this Application Note we have explored the use of Narrow Bore columns as well as a 10-port switching valve for the 1290 Infinity from Agilent to reduce the volume of solvent as well as the amount of substrate to be digested while at the same time allowing the process being automated and avoiding the contamination of the mass spectrometer with salt during the digestion process.
See Application Note 139:
Automated Digestion with StyrosZyme® TPCK-Trypsin, Immobilized Enzyme on Polymeric Hard Gel Simulated-Monolith™ Enzyme Reactor. Use of a 10 port valve to minimize salt exposure to the Mass. Spectrometer.