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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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Chromatograms obtained from injection of 10 pmol horse cytochrome C in capillary digestion systems monitored at 214 nm and 395 nm. The experiments were conducted with an APTES-CMD-AMD derivatised fused-silica capillary of (a) 510 × 0.050 mm, not containing trypsin, operated at 1 μL min−1 (blank); (b) 1400 × 0.050 mm, trypsin-modified, operated at 5 μL min−1 (average sample residence time 33 s); (c) as (b) but operated at 1 μL min−1 (average sample residence time 165 s). For clarity the beginning of the chromatogram displaying the 214 nm signal is offset as indicated
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Fig4: Chromatograms obtained from injection of 10 pmol horse cytochrome C in capillary digestion systems monitored at 214 nm and 395 nm. The experiments were conducted with an APTES-CMD-AMD derivatised fused-silica capillary of (a) 510 × 0.050 mm, not containing trypsin, operated at 1 μL min−1 (blank); (b) 1400 × 0.050 mm, trypsin-modified, operated at 5 μL min−1 (average sample residence time 33 s); (c) as (b) but operated at 1 μL min−1 (average sample residence time 165 s). For clarity the beginning of the chromatogram displaying the 214 nm signal is offset as indicated

Mentions: Chromatograms for the on-line digestions obtained with capillaries with an i.d. of 50 μm are shown in Fig. 4. Figure 4a shows a blank run in a capillary containing no enzyme and Fig. 4b and c present on-line digestions for a micro-reactor of 2.75 μL operated at 5 μL min−1 and 1 μL min−1, respectively. As discussed above, incomplete digestion of the injected cytochrome C will lead to the presence of multiple peptide fragments containing the heme group, as is visible in Fig. 4b. The digestion is complete when the sample exposure time is 165 s (flow rate 1 μL min−1) as both intermediate products and the undigested protein (retention time 44 min in Fig. 4a), which are visible at a wavelength of 395 nm as outlined above, are no longer observed. An injection of off-line-digested cytochrome C showed a similar chromatogram as is shown in Fig. 4c.Fig. 4


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)

Chromatograms obtained from injection of 10 pmol horse cytochrome C in capillary digestion systems monitored at 214 nm and 395 nm. The experiments were conducted with an APTES-CMD-AMD derivatised fused-silica capillary of (a) 510 × 0.050 mm, not containing trypsin, operated at 1 μL min−1 (blank); (b) 1400 × 0.050 mm, trypsin-modified, operated at 5 μL min−1 (average sample residence time 33 s); (c) as (b) but operated at 1 μL min−1 (average sample residence time 165 s). For clarity the beginning of the chromatogram displaying the 214 nm signal is offset as indicated
© Copyright Policy
Related In: Results  -  Collection

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

Fig4: Chromatograms obtained from injection of 10 pmol horse cytochrome C in capillary digestion systems monitored at 214 nm and 395 nm. The experiments were conducted with an APTES-CMD-AMD derivatised fused-silica capillary of (a) 510 × 0.050 mm, not containing trypsin, operated at 1 μL min−1 (blank); (b) 1400 × 0.050 mm, trypsin-modified, operated at 5 μL min−1 (average sample residence time 33 s); (c) as (b) but operated at 1 μL min−1 (average sample residence time 165 s). For clarity the beginning of the chromatogram displaying the 214 nm signal is offset as indicated
Mentions: Chromatograms for the on-line digestions obtained with capillaries with an i.d. of 50 μm are shown in Fig. 4. Figure 4a shows a blank run in a capillary containing no enzyme and Fig. 4b and c present on-line digestions for a micro-reactor of 2.75 μL operated at 5 μL min−1 and 1 μL min−1, respectively. As discussed above, incomplete digestion of the injected cytochrome C will lead to the presence of multiple peptide fragments containing the heme group, as is visible in Fig. 4b. The digestion is complete when the sample exposure time is 165 s (flow rate 1 μL min−1) as both intermediate products and the undigested protein (retention time 44 min in Fig. 4a), which are visible at a wavelength of 395 nm as outlined above, are no longer observed. An injection of off-line-digested cytochrome C showed a similar chromatogram as is shown in Fig. 4c.Fig. 4

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