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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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Results from on-line digestion of 10 pmol myoglobin at a flow rate of 1 μL min−1: (a) base peak chromatogram, the numbers correspond with the matched peptides in Table 3, and (b) the MS–MS spectrum of the peptide HGTVVLTALGGILK with m/z 689.7
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Fig6: Results from on-line digestion of 10 pmol myoglobin at a flow rate of 1 μL min−1: (a) base peak chromatogram, the numbers correspond with the matched peptides in Table 3, and (b) the MS–MS spectrum of the peptide HGTVVLTALGGILK with m/z 689.7

Mentions: In further experiments horse myoglobin was digested on-line. This protein is generally regarded as difficult to digest [15, 35]. When 1 μL of a 10 μmol L−1 solution in buffer is injected at a flow rate of 1 μL min−1 (165 s exposure time), the injected protein is completely digested, as was observed with UV detection (data not shown). Using mass spectrometric analysis, 13 different peptides are observed and matched using the MS–MS data and a Mascot database search, resulting in a sequence coverage of 88%. The matched peptides are summarized in Table 3 and a BPC of the on-line digestion of myoglobin is shown in Fig. 6a, with an MS–MS spectrum of one of the tryptic peptides. Both the degree of digestion and the sequence coverage are adequate compared with other systems that often use a high percentage of modifier to enhance digestion, as the absence of denaturing agents during digestion leads to little or no digestion of myoglobin [35]. Therefore these reactors are generally used for direct infusion into MS or off-line protein digestion as the presence of high concentrations of methanol or acetonitrile in the digestion buffer will seriously impede on-line protein digestion in combination with RP-LC.Fig. 6


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)

Results from on-line digestion of 10 pmol myoglobin at a flow rate of 1 μL min−1: (a) base peak chromatogram, the numbers correspond with the matched peptides in Table 3, and (b) the MS–MS spectrum of the peptide HGTVVLTALGGILK with m/z 689.7
© Copyright Policy
Related In: Results  -  Collection

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

Fig6: Results from on-line digestion of 10 pmol myoglobin at a flow rate of 1 μL min−1: (a) base peak chromatogram, the numbers correspond with the matched peptides in Table 3, and (b) the MS–MS spectrum of the peptide HGTVVLTALGGILK with m/z 689.7
Mentions: In further experiments horse myoglobin was digested on-line. This protein is generally regarded as difficult to digest [15, 35]. When 1 μL of a 10 μmol L−1 solution in buffer is injected at a flow rate of 1 μL min−1 (165 s exposure time), the injected protein is completely digested, as was observed with UV detection (data not shown). Using mass spectrometric analysis, 13 different peptides are observed and matched using the MS–MS data and a Mascot database search, resulting in a sequence coverage of 88%. The matched peptides are summarized in Table 3 and a BPC of the on-line digestion of myoglobin is shown in Fig. 6a, with an MS–MS spectrum of one of the tryptic peptides. Both the degree of digestion and the sequence coverage are adequate compared with other systems that often use a high percentage of modifier to enhance digestion, as the absence of denaturing agents during digestion leads to little or no digestion of myoglobin [35]. Therefore these reactors are generally used for direct infusion into MS or off-line protein digestion as the presence of high concentrations of methanol or acetonitrile in the digestion buffer will seriously impede on-line protein digestion in combination with RP-LC.Fig. 6

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