Precipitation peptides

The nitrogen content of this mixture amounted in the case of normal urine to 0.68-1.27% of the total nitrogen, whereas the peptides precipitated under these circumstances constituted 16.1-20.9% of all substances appearing in urine and giving the biuret reaction. The peptide mixtures obtained in identical conditions from six samples of pathological urine (liver cirrhosis and leukemia) exhibited marked differences in the nitrogen and peptide content as compared with those isolated from normal urine. 

Solid-phase synthesis was performed using Wang resin (0.57 mmol-g 1 0.35 g, 0.2 mmol) with the coupling of Fmoc amino acids and Fmoc-Hse[PO(OPh)2]-OH performed using PyBOP (3 mol equiv) and NMM (5 mol equiv) in DMF. The peptide-resin was treated with TFA/H20/TIS (95 2.5 2.5 5 mL) at rt for 2h, the resin filtered under N2, and washed with TFA (4mL). The solvent was removed under reduced pressure, the peptide precipitated by the addition of Et20 (20 mL), triturated with Et20 (2 x 10 mL), and dried under high vacuum. 

Dry 1/f-tetrazole (0.131 g, 1.875 mmol) was added to a suspension of Tyr-peptide-Wang-resin 44 (0.075 mmol) and (tBuO)2PNEt2 (0.187 g, 0.750 mmol) in anhyd DMF (0.5 mL) and the sintered reaction vessel rotated for 3 h under N2. The soln was removed under positive N2 pressure, the resin washed with CH2C12 (3 x 3mL) and treated with TFA/Et3SiH/PhOMe (95 2.5 2.5, 2mF) for 2h. The soln was collected, the solvent removed under reduced pressure and the peptide precipitated by the addition of Et20 (10 mL). The peptide was purified by RP-HPLC [0.1 M TEA (adjusted to pH 7.5 with AcOH), 0-40% MeCN over 40min, flow rate 2.0mL-min ] and gave the product as a white powder yield 41% MALDI-MS m/z 1992.5 [M + H],... 

At the end of the reaction time, filter the soln through a fritted funnel directly into cold, stirred Et20 or MTBE (200 mL). More complete precipitation of the peptide may be obtained if the Et20 is chilled in dry ice/EtOH. Wash the resin with three small portions of cold TFA. Add dry Py (1.0 mL) dropwise to the cold, stirred Et20 suspension to complete peptide precipitation. Collect the peptide on a fine-pore fritted glass funnel, wash with cold EtjO (3 X), and air-dry. Purify the crude peptide by an acceptable procedure. 

Acetone up to 80% vol/vol at 0 °C useful for peptide precipitation or concentration of sample for electrophoresis may denature protein irreversibly... 

Fill a 50mL conical tube about two-thirds full with cold diethyl ether. Add the concentrated peptide/TFA solution into cold ether using a Pasteur pipette. Place the cold ether with the peptide precipitate at -80°C freezer overnight. 

Centrifuge the cold ether with the peptide precipitate solution at 4°C for lOmin at 2,800-3,000 x f. Carefully decant the ether from the tube (see Note 23). 

Use peroxide-free ether for peptide precipitation and include EOT or EMS in cleavage cocktail. Ensure Met used is free of sulphoxides. Reduce sulphoxides using method in Chapter 4, Protocol 1... 

TFA and DCM can be removed under a stream of N2 and the peptide precipitated with diethyl ether, centrifuged before the diethyl ether is removed. The ether treatment is repeated. 

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