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Development of an open-tubular trypsin reactor for on-line digestion of proteins.

Stigter EC, de Jong GJ, van Bennekom WP - Anal Bioanal Chem (2007)

Bottom Line: Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 degrees C and are stable for at least two weeks when used continuously.Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures.These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Analysis, Department of Pharmaceutical Sciences, Faculty of Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA, Utrecht, The Netherlands. e.c.a.stigter@uu.nl

ABSTRACT
A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm(-2) trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 degrees C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol microL(-1) horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm x 50 microm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening.

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Set-up used for on-line protein digestion using a trypsin-modified fused-silica capillary. For detection a PDA or an ion-trap MS was used
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Fig2: Set-up used for on-line protein digestion using a trypsin-modified fused-silica capillary. For detection a PDA or an ion-trap MS was used

Mentions: For the on-line peptide and protein digestion experiments the trypsin-modified capillaries were positioned between the LC injector and a 10-port valve, as shown in Fig. 2, and were immersed in a thermostatted water bath set at 37 °C unless mentioned otherwise. Protein samples were prepared in digestion buffer and were transported through the capillary towards a 10-port valve using a 5% acetonitrile solution containing 0.05% TFA. The peptide fragments formed during digestion were concentrated on an RP trapping column (TC) and salts and other buffer components present in the sample were removed. By switching the valve the trapping column is in series with the RP micro column and an acetonitrile gradient started. The gradient was composed of two solutions: (A) 5% acetonitrile in water containing 0.05% TFA and (B) 80% acetonitrile in water containing 0.04% TFA. In 30 min the gradient changed linearly from 0 to 50% B, followed by 10 min at 90% B and 20 min at 0% B. The eluent was monitored with the PDA detector in the range from 200 to 595 nm.Fig. 2


Development of an open-tubular trypsin reactor for on-line digestion of proteins.

Stigter EC, de Jong GJ, van Bennekom WP - Anal Bioanal Chem (2007)

Set-up used for on-line protein digestion using a trypsin-modified fused-silica capillary. For detection a PDA or an ion-trap MS was used
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2117336&req=5

Fig2: Set-up used for on-line protein digestion using a trypsin-modified fused-silica capillary. For detection a PDA or an ion-trap MS was used
Mentions: For the on-line peptide and protein digestion experiments the trypsin-modified capillaries were positioned between the LC injector and a 10-port valve, as shown in Fig. 2, and were immersed in a thermostatted water bath set at 37 °C unless mentioned otherwise. Protein samples were prepared in digestion buffer and were transported through the capillary towards a 10-port valve using a 5% acetonitrile solution containing 0.05% TFA. The peptide fragments formed during digestion were concentrated on an RP trapping column (TC) and salts and other buffer components present in the sample were removed. By switching the valve the trapping column is in series with the RP micro column and an acetonitrile gradient started. The gradient was composed of two solutions: (A) 5% acetonitrile in water containing 0.05% TFA and (B) 80% acetonitrile in water containing 0.04% TFA. In 30 min the gradient changed linearly from 0 to 50% B, followed by 10 min at 90% B and 20 min at 0% B. The eluent was monitored with the PDA detector in the range from 200 to 595 nm.Fig. 2

Bottom Line: Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 degrees C and are stable for at least two weeks when used continuously.Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures.These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening.

View Article: PubMed Central - PubMed

Affiliation: Division of Biomedical Analysis, Department of Pharmaceutical Sciences, Faculty of Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA, Utrecht, The Netherlands. e.c.a.stigter@uu.nl

ABSTRACT
A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm(-2) trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 degrees C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol microL(-1) horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm x 50 microm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening.

Show MeSH
Related in: MedlinePlus