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On-Bead Digestion Protocol
N.B. The protocol here is for magnetic beads, but with adjustments for volume, could also be used for
agarose beads too.
Material List
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Eppendorf Protein Lobind tubes (Sigma Z666505 or equivalent, not strictly required, but
preferred for final elution)
Ammonium Bicarbonate (Sigma A6141 or equivalent)
DL-Dithiothreitol (Sigma D0632 or equivalent)
Iodoacetamide (Sigma I1149, bulk, A3221 pre-weighed, or equivalent)
Trypsin (Sequencing Grade, Promega V5111 … not the pronounced lack of “or equivalent”)
Formic Acid (ACS Grade or higher, Sigma F0507, or equivalent)
Trifluoroacetic acid (ACS Grade or higher, Sigma T6508)
Water (HPLC Grade or higher)
Acetonitrile (HPLC Grade or higher)
C18 Spin Columns (ThermoPierce #89870)
Protocol
For all steps below, 100 uL has been chosen as the volume of choice. This is not critical. The important
thing is that the beads are covered with solution.
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Perform upstream steps to obtain protein bound to beads.
Wash beads in 100mM ammonium bicarbonate
Remove supernatant.
Reduction: Add 100uL of 10 mM DTT (dithiothreitol, Sigma D0632 or equivalent) in 100mM
ammonium bicarbonate. Incubate at 50 °C for 30 minutes (with gentle shaking/rocking,
optional).
Cool to room temperature. Isolate beads and discard supernatant.
Alkylation: Add 100uL of 100mM IAA (iodoacetamide, freshly made, Sigma A3221) in 100mM
ammonium bicarbonate. Incubate *in the dark* at room temperature for 30 minutes (with
gentle shaking/rocking, optional).
Isolate beads and discard supernatant.
Wash beads with 100uL of 100mM ammonium bicarbonate
Trypsin Digestion: Resuspend beads in 100 uL 100mM ammonium bicarbonate. Add ~0.5-1ug
trypsin (Promega V5111), incubate at 37°C overnight (8-16 hours).
Stop trypsin digestion by adding 2uL formic acid.
Isolate beads and collect supernatant containing peptides for next step.
Perform Peptide Isolate/Recovery: Follow protocol for C18 spin column
http://www.piercenet.com/product/c18-spin-columns