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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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Related in: MedlinePlus

On-line digestion of 10 pmol cytochrome C at a flow rate of 1 μL min−1: (a) base peak chromatogram, the numbers correspond to the matched peptides in Table 3, and (b) MS–MS fragmentation of the peptide TGPNLHGLFGR with m/z 584.9 showing several of the matched fragment ions
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Fig5: On-line digestion of 10 pmol cytochrome C at a flow rate of 1 μL min−1: (a) base peak chromatogram, the numbers correspond to the matched peptides in Table 3, and (b) MS–MS fragmentation of the peptide TGPNLHGLFGR with m/z 584.9 showing several of the matched fragment ions

Mentions: The on-line digestion of horse cytochrome C is also monitored with mass spectrometry. The effect of flow rate and hence incubation time on the digestion of the protein and the number of peptides identified with a Mascot database search is determined and summarized in Table 2. In this table the undigested amounts of protein which have been determined using the PDA detector are also shown. At a flow rate of 1 μL min−1 protein digestion is complete and many peptides are matched, resulting in high sequence coverage and Mascot score. With higher flow, and hence decreasing incubation time, the amount of protein that remains undigested increases and consequently fewer peptides are produced and observed. However, even at relatively high flow rates an adequate amount of peptides is still formed and the protein can be identified on the basis of the fragments present. Nevertheless, the ion intensity is low and some peptides are not retrieved as they are below the threshold for auto-MS–MS. Table 3 summarizes the peptides observed and matched using the MS–MS data and a Mascot.database search of proteins digested at a flow rate of 1 μL min−1. A base-peak chromatogram (BPC) of the digestion of cytochrome C under these conditions is shown in Fig. 5a. The MS–MS fragmentation of one of the peptides is presented in Fig. 5b.Table 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)

On-line digestion of 10 pmol cytochrome C at a flow rate of 1 μL min−1: (a) base peak chromatogram, the numbers correspond to the matched peptides in Table 3, and (b) MS–MS fragmentation of the peptide TGPNLHGLFGR with m/z 584.9 showing several of the matched fragment ions
© Copyright Policy
Related In: Results  -  Collection

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

Fig5: On-line digestion of 10 pmol cytochrome C at a flow rate of 1 μL min−1: (a) base peak chromatogram, the numbers correspond to the matched peptides in Table 3, and (b) MS–MS fragmentation of the peptide TGPNLHGLFGR with m/z 584.9 showing several of the matched fragment ions
Mentions: The on-line digestion of horse cytochrome C is also monitored with mass spectrometry. The effect of flow rate and hence incubation time on the digestion of the protein and the number of peptides identified with a Mascot database search is determined and summarized in Table 2. In this table the undigested amounts of protein which have been determined using the PDA detector are also shown. At a flow rate of 1 μL min−1 protein digestion is complete and many peptides are matched, resulting in high sequence coverage and Mascot score. With higher flow, and hence decreasing incubation time, the amount of protein that remains undigested increases and consequently fewer peptides are produced and observed. However, even at relatively high flow rates an adequate amount of peptides is still formed and the protein can be identified on the basis of the fragments present. Nevertheless, the ion intensity is low and some peptides are not retrieved as they are below the threshold for auto-MS–MS. Table 3 summarizes the peptides observed and matched using the MS–MS data and a Mascot.database search of proteins digested at a flow rate of 1 μL min−1. A base-peak chromatogram (BPC) of the digestion of cytochrome C under these conditions is shown in Fig. 5a. The MS–MS fragmentation of one of the peptides is presented in Fig. 5b.Table 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