Membrane Protein Digestion

From Mswiki

Membrane Protein Digestion with High pH and Proteinase K

1. Pellet membranes out of whatever buffer they were prepared and isolated in. Microfuge at 14,000 RPM for 20 min at 4ºC. Discard supernatant. ALTERNATIVELY, if your sample is already in solution, adjust the pH to 11 with 1 M NaOH and skip to step 3.

2. Resuspend membrane pellet in high pH buffer (0.2 M Na2CO3, pH 11) at a concentration of ~1mg/ml using 5 strokes through an insulin syringe or tissue grinder/homogenizer.

3. Incubate on ice for 1 hour.

4. Add solid urea to 8M (0.48 g/ml). Reduce and alkylate with DTT/IAA or TCEP/IAA.

5. Add proteinase K at 1/100 enzyme/substrate ratio and incubate at 37ºC for 3 hours. Add another aliquot of proteinase K (final ratio is 1/50) and incubate at 37ºC for 1.5 hours.

6. To store: Freeze in –20/-80 to stop reaction.

7. To analyze: Add formic acid to 5%. There will be a little bubbling as the high pH is neutralized so leave the caps open until the reaction is complete. Microfuge to pellet out membranes at 14,000 RPM for 20 min at 4ºC. Take off supernatant and load onto a split column packed with reverse phase (AQUA or Polaris A).

Notes by McClatchy 1. pK at a ratio of 1/50 for 5 hours works the same as protocol above. 2. A MudPIT with a shallow buffer B gradient works the best for identifications, since the peptides tend to be very similar. I usually use a gradient of 10% to 30%.