OraChrom, Inc.

APPLICATION NOTE

AN070802-26

StyrosZymeä TPCK-Trypsin, Immobilized Enzyme on Polymeric Hard Gel Stationary Phase: Online Digestion of Lysozyme in 5 minutes.

Automation has always been the ultimate goal in different attempts by manufacturers of immobilized enzyme.

Soft gel media such as Agarose have been used to effectively reduce the auto digestion. They are not however, an appropriate stationary phase for packing small bore columns that can withstand high backpressures. Silica as another alternative, is not stable at high pH’s required for protein digestions.

Hard gel, fully pervious polymeric such as STYROS™ media, combine soft gel’s high surface and silica’s high rigidity.

STYROS™ is made of highly crosslinked polystyrene-divinyl benzene Gigaporous matrix that can withstand backpressures of up to 10,000 psi, as well as extremes of pH’s. The chemical and mechanical stability provides the optimum support for the full range of immobilized components such as enzyme.

The present application note shows the practical aspect of the resulting product commercialized by OraChrom under the name StyrosZyme™ TPCK-Trypsin.

The present set up consists of the StyrosZyme™ column (2.1 x 50 mm) connected in tandem with STYROS™ 2R/XH (4.6 x 250 mm), also reversed phase polymeric stationary phase that can withstand the same high pH’s used during protein digestion.

A known amount of Cytochrome c is injected into the StyrosZyme™ column at 100 ml/min of flow rate for 2 minutes. The flow rate is stopped for another 2 minutes. The digest is then flushed into the reversed phase column at 1 ml/min for 1 minute. After switching the enzyme column off line, the reversed phase column is equilibrated with 97 % buffer A, 3 % buffer B at 3 ml/min flow rate until stable baseline. The flow rate is then reduced to 1 ml/min and the resulting peptides are mapped following the indicated gradient.

The enzyme digest can alternatively be injected into a mass spectroscopy chamber either directly or after a preliminary separation on the reversed phase column. The possibility of using mass friendly buffer components with polymeric stationary phases does facilitate the LC-MS hyphenation.

Table 1. Operating parameters for the chromatograms.

HPLC System.	HP 1100
Columns	StyrosZymeä TPCK-Trypsin 2.1 x 50 mm STYROSä 2R/XH 4.6 X 250 mm
Mobile Phase For reversed phase.	A: 0.1% NH4OH in H2O B: 0.1% NH4OH in ACN:H2O (95:5)
Mobile Phase For Digestion.	3 % ACN in 50 mM CO3HNH4 and CO3(NH4)2 aqueous buffer at pH=7.8 and 10 respectively..
Flow rate	As indicated.
Gradient	7 to 34 % B in 27 min.
Temperature	37°C
Detection	214 nm
Injection volume	10 ml
Sample:	10 mg/ml Lysozyme in 0.1 % TFA.

The following two chromatograms depict the effect of pH on the digestion.

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APPLICATION NOTE

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