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StyrosZyme™ TPCK-Trypsin: digestion at different pH.
The protein is digested during a 5 min run in a 2.1 x 50 mm immobilized TPCK-Trypsin column. The digests are then deposited on a STYROS™ 2R/XH (4.6 X 250 mm) polymeric column and subsequently mapped.

Digestion at pH = 10 Figure 2. Peptide map of Cytochrome c digest on a STYROSä 2R/XH 4.6 x 250 mm reversed phase column.

Contents:

• Product Description
• Overview of On-line Digestion
• General Considerations

• Product Description: 

StyrosZymeä TPCK-Trypsin is a monolithic, fully uniform hydrophilic polymer based packing with TPCK-Trypsin covalently tethered to the surface of a Simulated Monolith Bed.
It is intended to perform online digestion of protein in a flow through setting.

The base matrix that is fully pervious, is made of highly crosslinked poly(styrene-divinylbenzene).

Our proprietary polymerization technique ensures that the beads are not brittle and as a result, free of any leachables. The unique macroporous structure takes full advantage of the inner bead’s surface area, making it possible to run high speed, high resolution separations without any mass transfer restrictions.

The polystyrenic matrix is rendered hydrophilic through a covalently bound coating.

The packed cartridges offer extremely high-pressure tolerance, and can be used routinely up to 4,000 psi.

The advantages offered by immobilized enzyme in a flow through setting are numerous:

·         Digestion time is reduced to a few minutes as compared to hours.

·         The enzyme cartridge can be used as a direct inlet to either an LC or an MS system for the analysis of the resulting peptides, substantially reducing and simplifying the sample handling process and allowing it to be fully automated.

·         The extent of digestion can be controlled by changing the flow rate and the temperature. It can also be made fully reproducible.

·         The immobilized enzyme displays high stability towards pH’s, organic solvents, high flow rates, temperatures and back pressures.

·         The possibility of using fast flow rates allows the cartridge to be reconditioned quickly, further reducing the process time.

·         Due to the absence of contact in the immobilized format between enzyme molecules, no auto digestion occurs.

StyrosZymeä Trypsin cartridges are offered in a number of formats to provide a wider selection

• Technical Specifications

• Overview of On-line Digestion:

StyrosZymeä columns can be used in series with a reversed phase column followed by a mass spectrometer or as a direct inlet to an MS.

Keep in mind that StyrosZymeäcolumns can tolerate back pressures of up to 4,000 psi. It is therefore possible to equilibrate and clean them in a very short period of time by running high flow rates.

 The digestion-mapping process involves the following steps:

·        Equilibration: Each column is equilibrated with the appropriate buffer solutions.

·       Loading: The protein of interest is injected onto the StyrosZymeä enzyme reactor column.

·      Digestion: Using the optimum residency time and temperature, the protein is fully digested on-line. The resulting peptides are dumped on the reversed phase column.

·        Separation: The hydrophobically bound peptides are then eluted from the reversed phase column with the option of being further introduced to an MS.

·        Cleanup and re-equilibration: Each column is then eluted with the appropriate buffer.

• General Considerations:

Enzyme digestion varies with proteins as well as the operating conditions such as temperature, flow rate and pH.

By controlling these variables, it is possible to control the extent of digestion. The size of the peptide fragments changes with the temperature as well as the flow rate.

At pH 7-8, Trypsin cleaves the peptide bonds of the protein at the carboxy terminus of arginine, lysine, and S-2-aminoethylcysteine residues.

At pH 11 the cleavage is more selective to lysyl residues in the presence of S-2-aminoethylcysteine.

The tandem reversed phase column in such set up needs to tolerate high pH’s. Polymeric reversed phase columns such as STYROSä 2R/XH are highly recommended, especially when the effluents are used to feed a mass spectrometer.

The presence of some denaturing agents such as organic solvents, TFA or 3M guanidine HCl promotes the digestion. The proteins that are difficult to digest in solution will be difficult to digest on line. It is necessary to assist the unfolding of the protein by denaturing agents and prevent the refolding by using reducing or alkylating agents.

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