BAF_Protocol_002 On-Bead Digestion (Magnetic Beads) of Proteins in Solution or Provided Already on Beads
Nicholas Sherman
Abstract
This protocol is used to more easily digest a tissue or cell protein lysate (after precipitation and reconstitution) on a magnetic bead for easier manipulation and washing. The protocol from step 13 on would be used for proteins already captured on a magnetic beads such as antibody (IP) or avidin coated - usually as an enrichment step. The proteins are easily washed to remove contaminants such as detergents or salts. The resulting peptide mixture can be further purified using a C18 tip such as our Protocol 003.
Before start
REAGENTS: (All reagents to be prepared fresh for each digestion)
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100 mM ammonium bicarbonate (ABC): 0.158 g in 20 mL distilled water
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50 mM ABC: 0.079g in 20 mL distilled water
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Acetonitrile (ACN)
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Ethanol
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100 mM DTT: 0.0015g in 100 uLof 100mM ABC (DO NOT mix until directly before you are ready to use)
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500 mM Iodoacetamide: 0.01g in 100 uL of 100 mM ABC (DO NOT mix until directly before you are ready to use)
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Trypsin solution: Keep on ice. Promega (cat. # V5113) is already diluted in 50 mM acetic acid.
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DMSO 2% in water.
Steps
Prepare Stock Solution of Beads (for Tissue or Cell Lysates)
Remove both beads (A and B) from the fridge and keep them at room temperature for 10 minutes. Combine 20 uL of magnetic beads A and 20 uL of magnetic beads B.-
Add 160 uL of water (LC-MS/MS grade).
Place tubes with beads on a magnetic rack and let beads settle for 2 minutes. Remove and discard supernatant.
Rinse beads with 200 uL of water by pipette mixing (off the magnetic rack). Place tubes with beads on a magnetic rack and let beads settle for 2 minutes. Remove and discard supernatant.
Repeat step 4 two times.
Add 100 uL of water to beads, 10ug/uL stock bead mix, and store in the fridge. Prepared beads can be stored at 4ºC indefinitely. Never freeze the beads.
On-bead Digestion (Start Step #13 if Beads were Affinity Capture)
To 10 ug of total protein add 2 uL of bead mix (20 ug) from step 6 in the total volume of 200 uL FA 0.1% (aim for a pH ~2).
Add 200 uL of ACN (100% stock) to reach a final concentration of 50% (v/v). Incubate for 8 minutes at room temperature.
Place the tubes on a magnetic rack for 2 minutes. Remove and discard supernatant.
While on the magnet rack, rinse beads with 200 μl of 70% absolute ethanol, incubate for 30 s, and discard supernatant.
Repeat step 10 once.
Rinse beads with 180 μl of 100% ACN, incubate for 30 s, and discard supernatant.
Add 20 uL of 100 mM ABC buffer. If using beads that already have proteins captured such as IP (antibody), Biotin, etc, wash the beads three times with 200 ul ABC buffer to remove previous buffer and then add 20 uL ABC.
Reduce the proteins by adding 2 μL of 100 mM DTT to the beads, incubate for 30min at room temperature.
Alkylate the proteins by adding 2 μL 500 mM iodoacetamide at room temperature for 30 min in dark.
Dilute the sample with 20 µL of water (final ABC concentration to 50 mM). Check the sample pH it should be around 8-7.
Add 2.5-5 μL of Promega trypsin on ice to each sample for about 1 µg of trypsin.
Incubate overnight at 37ºC, 700 rpm.
Add 1000 uL of ACN 100% to final concentration > 95%. Incubate for 8 minutes at room temperature.
Place the tubes on a magnetic rack for 2 minutes. Remove and discard supernatant.
Rinse beads with 180 μl of 100% ACN, incubate for 30 s, and discard supernatant.
Add 10 uL of 2% DMSO (in water, not acidic). Give a quick spin (~2 seconds) in a bench-top centrifuge to aid the liquid removal from the tube walls.
Place tubes on a magnetic rack for 2 minutes and recover supernatant making sure to not recover any beads.
Dilute the samples with 0.1% FA to <1% DMSO and go to BAF_Protocol_003 - Dessalting with C-18 tips.
