APPLICATION NOTE

AN032505-28

The chemical and mechanical stability of polymeric hard gel media that are the features of StyrosZymeä immobilized enzyme columns, allow their use on line in a flow through setting.

The advantages resulting from such 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 a LC or a 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 low pH’s, 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.

·         No autodigestion occurs due to the absence of contact between enzyme molecules in the immobilized format.

·         A single cartridge can be used during many digestions without loosing its activity.

·         The ratio of Enzyme to Substrate can be controlled in order to obtain target peptide in different concentrations.

In the following examples Cytochrome c from two different sources were used to highlight the variations in peptides profiles obtained during reversed phase mapping of the resulting digests.

Similar conditions are used in both cases to achieve over 99% digestion.

A typical digestion consists of running a known amount of protein in the StyrosZymeä Pepsin column at flow rates of 50 µl/min (18 cm/hr for a 4.6 mm ID column). The resulting peptide digests are dumped into a reversed phase column connected in series with the StyrosZymeä Pepsin column.

The enzyme column is then taken off line and the peptide mapped following a gradient.

Chromatogram 1

Peptide digests from Cytochrome c from horse heart separated on a STYROS™ 2R/XH 4.6 X 150 mm at 0.5 ml/min (180 cm/hr)

Chromatogram 2

Peptide digests from Cytochrome c from bovine heart separated on a STYROS™ 2R/XH 4.6 X 150 mm at 0.5 ml/min (180 cm/hr)

Table 1. Operating parameters for the chromatograms.

	HPLC System	Agilent 1100
	Detector	214 nm
	Column	StyrosZyme™ Pepsin 33 x 4.6 mm STYROS™ 2R/XH 4.6 x 150 mm
	Mobile Phase for Digestion	100 mM AcONa + 150 mM NaCl in DI H2O at pH=4.5.
	Mobile Phase for Separation	A: 0.075 % TFA in H2O B: 0.075 % TFA in ACN:H2O (95:5)
	Gradient	As indicated
	Flow rate	As indicated
	Temperature	48 °C
	Injection volume	10 µl
	Sample:	10 mg/ml Cytochrome c in mobile phase buffer A

 

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