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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 notes describe the details of the automated digestions on HPLC instruments.
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.
A Narrow Bore enzyme column of 2.1×100 mm stainless Steel (StyrosZyme® TPCK-Trypsin) as well as a reversed phase Narrow Bore column (STYROS® 1R) column of the same size are used with Waters Acquity UPLC I class Plus with a 2 positions 6-port switching valve.
We have used the insulin B Chain to run the digestion.
The linearity of the peak height as well as the peak area were checked to verify the reproducibility of the digestion run at high pH’s of 8.5.
See Application Note 140:
Two 6 ports, two positions switching valve are used with the Acquity UPLC I class Plus in order to ultimately map the peptides resulting from the online digestion of Lysozyme on a Silica C18 column (Acquity UPLC® BEH C18 1.7 µm 2.1×50 cm column).
See Application Note 141:
Using the same setup as in the previous Application Note 141, Trypsin itself was used for the digestion.
See Application Note 142:
With the same setup as in the previous Application Note 142, two types of Cytochrome c are digested and compared with one other.
See Application Note 143:
Comparing digests of Cytochrome c from Bovine with Equine with StyrosZyme® TPCK-Trypsin, Immobilized Enzyme on Polymeric Hard Gel Simulated-Monolith™ Enzyme Reactor with the Acquity UPLC I class Plus and Final mapping on Silica C18. Fully Automated..
In addition of being tedious and lengthy, the process of batch digestion of proteinaceous species is also froth with the contamination of enzymes auto digests.
It is well understood that as the digestion proceeds in batch, the auto-digested enzymes no longer have a similar behavior as the intact enzymes.
Moreover, batch digestion is an anachronistic process that should no longer remain in the lexicon of today’s concept of real-time modus operandi.
To highlight the point, we have plotted the previously digested Trypsin on the immobilized Trypsin reactor, along with the digests of Cytochrome c from equine using similar conditions in both cases.
See Application Note 144: