Endoproteinases Asp

Fig. 5 Screening by affinity CE for interaction of SAP peptides with heparin in solution. An endoproteinase Asp-N-treated Glu-C digest of SAP solubilized in water was injected for 12 s and subjected to CE at 15 kV (detection at 200 nm) in the presence of heparin (Hep) (B) added to the electrophoresis buffer (0.1 M phosphate, pH 7.5) at the concentration indicated. The peptide marked with asterisks was identified by spiking with HPLC-purified fragments and corresponds to the fragment in Figure 6. (From Ref. 71.)...

Figure 1. Endoproteinase Asp-N peptide mapping of r-HuB61 24KD (chromatogram A) and 22 (chromatogram B) forms.

All chemicals used were of analytical grade, except when otherwise indicated. Endoproteinase Asp-N was obtained from Boehringer Mannheim (Indianapolis, IN). labeled recombinant human leptin was expressed in E. coli in inclusion bodies using [15N]ammonium sulfate as the sole nitrogen source. The protein was allowed to fold and oxidize after solubilization of inclusion bodies, and puriHed by ion exchange chromatography to 95% purity as assessed by SDS-polyacrylamide gel electrophoresis as described (9, 11). 

The solution of [15N]r-metHuLeptin (0.3 mg/ml) in PBS (0.1 M sodium phosphate, 0.1 M sodium chloride, pH 7.2) was incubated with endoproteinase Asp-N at an enzyme-to-substrate ratio of 1 75 (w/w) at 25 °C for 5 h. The digestion was terminated by adding 5 )tL of 5% TFA to the reaction. Peptides were separated by a Vydac C4 reverse-phase analytical column (4.6x250 mm) using a Hewlett Packard HPLC (Model 1090), which is on-line connected to a PE-Sciex API-100 electrospray mass spectrometer. The column was initially equilibrated with 95% mobile phase A (0.1%TFA) and 5% mobile phase B (90% acetonitrile in 0.1% TFA). A linear gradient from 10 to 50% mobUe phase B was run over a period of 85 minutes at a flow rate of 0.5 raL/min. The splitting of the flow (9 1) was achieved post UV cell, allowing 50 iL/min of the eluent to be analyzed by the electrospray mass spectrometer. 

Endoproteinase Asp-N Origin Pseudomonas fragi (mutant) Roche Diagnostics Endoproteinase Asp-N, Sequencing Grade... 

Example 8.2 A peptide of sequence Tyr-Phe-Ser-Asp-Phe-Leu-Arg-Trp-Val-Gly-Met-Glu-Ala-Asp-Phe-Gly-Thr-Lys-Ser-Asn-Ala-Leu is treated first with endoproteinase Asp-N and then with trypsin. How many peptides will be generated by this treatment, and what will be each peptide s molecular weight ... 

In another forensic science application, electrospray LC-MS was used to determine elevated levels of insulin-like growth factor 1 (IGF-1) in serum as an indication of growth hormone (GH, somatotropin) abuse in racehorses. Structural identity of IGF-1 was confirmed by LC-MS and LC-MS/MS characterization of the endoproteinase Asp-N digestion product. 

Enzyme stock preparations To make 1 ml trypsin, chymotrypsin, or endoproteinase Glu C stock solution, dissolve 1 mg enzyme in 1 ml UHQ water. Distribute the solution in 50-//1 portions in 0.5-ml Eppendorf tubes, lyophilize in a vacuum centrifuge, and store at —20°C. To make 0.2 ml endoproteinase Asp-N stock solution, dissolve 10 //g enzyme in 200 //I UHQ water. Distribute in 10-/xl portions, lyophilize, and store as above. To make 100 //I carboxypeptidase Y, A, or M stock solution, dissolve 100 //g enzyme in 100 //I UHQ water. Distribute in 10-(A portions, lyophilize, and store as above. 

Distribute 5 1 of reduction buffer or enzyme solution on the surface with an automat pipette and place a microscope cover glass on top of the target. To make the enzyme solution, redissolve an aliquot of the lyophilized stock solution to a concentration of 1 figlpd (for endoproteinase Asp-N 0.5 /xg/Atl) in 0.1 M ammonium bicarbonate solution adjusted to the desired pH (for digestion with endoproteinases, pH 7.8 and, for carboxypeptidases, pH 4.2 are generally used). 

If aggregation of proteins occurs, change the dialysis buffer to 2 taM Tris-HCl Chelator molecules such as EDTA should be completely removed, because endoproteinase Asp-N is a Zn-proteinase The dialysate can be stored at 4°C Add 2ME for long-term storage. 

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