Protein collection and prep for proteomics (Ostracods)

Materials

• 100% Trichloroacetic acid
• 0.01 M HCl
• Acetone
• 100 mM TRIC-HCl (pH 8.5)
• 8M Urea
• 100 mM TCEP
• 500 mM Iodoacetamide (made fresh)
• Trypsin (0.5 ug/ul)
• Formic Acid
• Methanol
• Acetonitrile
• C18 Spec Tips (Agilent cat#A57203)

Protein collection

1. Place ostracods in collection chamber (~3cm of aquarium tubing filled with sea water)
2. Shock for 10secs with 12V or until bioluminescent response is elicited (note: do not shock continuously for over 10secs, instead use short shocking pulses)
3. Remove fluid using a filter pipette and place into sterile 1.5mL tube. If possible, wear gloves and clean work station of dust or other contaminants.

Protein precipitation

1. Add 100% trichloroacetic acid (TCA) to bring solution to 20% TCA
2. Leave at -20*C overnight
3. Spin down at max speed for 10min at 4*C
4. Wash (x3 times) with an ice cold solution of 10% 0.01 M HCl and 90% acetone
5. Air dry pellet for 10min (usually)
6. Resuspend in ~80ul of 50% 100mM TRIS (pH 8.5), 50% 8M Urea

Protein digestion=

1. Add 100mM TCEP to a final concentration of 5 mM. Incubate at room temperature for 5min.
2. Add 500 mM Iodoacetamide to a final concentration of 10 mM. Make fresh each time. Incubate at room temperature for 15min in the dark.
3. Dilute samples by a factor of four with 100 mM TRIS-HCl (pH 8.5)
4. Add 100 mM CaCl2 to final concentration of 1 mM
5. Add 1ul (0.5 ug/ul) trypsin
6. Incubate overnight at 37*C in the dark
7. Add formic acid to 5% final concentration (use glass to pipette concentrated acid)

Protein desalting

1. Wet C18 Spec tip with 200ul HPLC grade methanol
2. Wash (x3 times) tip with 200 ul of 5% acetonitrile/ 5% formic acid solution
3. Push sample through the tip
4. Wash tip with 200 ul of 5% acetonitrile/ 5% formic acid solution
5. Push sample through again
6. Wash (x3 times) tip with 200 ul of 5% acetonitrile/ 5% formic acid solution
7. Elute (x2 times) with 100ul of 80% acetonitrile/ 5% formic acid solution
8. Speed vacuum to dryness (Mazer lab has one that works)