APPLICATION NOTE

AN073102-27

StyrosZyme™ TPCK-Trypsin, Immobilized Enzyme on Polymeric Hard Gel Stationary Phase: Online Digestion of Insulin Oxidized B-chain in 16 minutes.

The active form of insulin consists of 2 chains connected by disulfide bonds between cysteine side chains.

Oxidized B-Chain consists of the following amino-acid sequence:

Phe-Val-Asn-Gln-His-Leu-Cys-(SO3H)-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-(SO3H)-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala

A typical digestion in solution with high grade Trypsin, requires a minimum of 18 hrs at 37 °C.

StyrosZyme™ TPCK-Trypsin can achieve the same result on line, in 16 minutes at 37 °C.

The resulting peptides can be mapped directly after digestion by using STYROS™ 2R reversed phase column connected in series to the immobilized enzyme column in less than 3 minutes.
 

The set up consists of a StyrosZyme™ TPCK-Trypsin column (2.1 x 100 mm), in tandem with the reversed phase polymeric STYROS™ 2R/XH (4.6 X 150 mm) column, capable to withstand long term high pH’s.

A known amount of Insulin Oxidized B-chain in solution is injected into the immobilized enzyme column and run for 14 minutes at 25 ml/minute, followed by 2 minutes at 1 ml/min. The effluent from the StyrosZyme™ TPCK-Trypsin column are directly deposited onto the polymeric STYROS™ 2R/XH column. The enzyme column is then switched off line. The reversed phase column is equilibrated and the resulting peptides are mapped by developing a gradient as indicated on the chromatograms. The entire digestion and mapping can be automated and run in less than 30 minutes including the re-equilibration of both columns.

Slower flow rates minimally affect the resolution as shown in chromatogram 2.

 Table 1. Operating parameters for the chromatograms.

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