Immobilization of Pronase on Sepharose Beads and Digestion of Glycopeptides_v1

Analytical Glycomics Laboratory Office (530) 752-5504 SOP: Procedure Study Revision Date: 3/3/2014, edited by Lauren
Procedure: Immobilization of Pronase on Sepharose Beads and Digestion of Glycopeptides
Purpose: To cleave peptide bonds and form glycopeptides. Trainer: Charles Nwosu
Author: Charles Nwosu, edited by Lauren
Supplies and samples required:
∙ CNBr activated sepharose 4B beads (GE Healthcare) –Sigma #C9142-5G
∙1mM HCl
∙Phosphate buffer solution (0.1M, Sigma, #P5244) pH 7.5∙Pronase E (Sigma- Protease, type XIV #P5147-1G)
∙1M aqueous ethanolamine - pH 9.0
∙Microcentrifuge (low speed to gently centrifuge beads to bottom of vial)
∙Gel Tips
∙0.05% (w/w) NaN3 in 100mM NH4OAc(optional –only for storage)
PPE: Lab coat and gloves at all times.
Instrument(s) that will be used to run samples:
∙Chip/TOF, Chip/Q-TOF
∙QQQ
Methods:
1.Weigh 150mg CNBr activated sepharose 4B beads into a
1.5 mL microfuge tube
2.Add 1 mL of 1mM HCl to suspend the beads. Agitate
gently for approximately 30 min.
3.Briefly centrifuge the suspension using a microfuge to
separate beads from solution.
4.Remove and discard the supernatant.
5.Wash the beads with 500uL of phosphate buffer (pH 7.4).
Repeat, for a total of three washings (3 cycles).
6.Prepare Pronase E solution:
a.Weigh 1mg of Pronase E.
b.Add 100uL 50:50 Phosphate buffer:H2O.
7.Add 500uL of fresh phosphate buffer and 100uL of the
prepared Pronase E solution to the sepharose bead suspension.
8.Allow the pronase bead coupling to continue overnight
(or for 2 hours) with gentle agitation at room temperature. This ensures quantitative covalent coupling of proteases to the beads via reaction of 1˚amino groups with the CNBr activated sepharose support.
9.Briefly centrifuge the suspension using a
microcentrifuge. Remove and discard the supernatant. 10.Wash the beads 3-5 times (using a very low vortexing
speed, or just with repeated inversion) with 500uL of phosphate buffer to remove excess enzymes.
11.After removing the last wash supernatant, add 500uL of
1M aqueous ethanolamine (pH 9.0) to block all remaining active sites of the beads.
12.Allow blocking of residual active sites to proceed for 2
hours with gentle agitation at room temperature.
13.Centrifuge the suspension using a microcentrifuge for ~1
minute, and remove the supernatant.
14.Wash the beads with 500uL of phosphate buffer. Repeat,
for a total of 4 washings, to remove residual ethanolamine.
15.(OPTIONAL–for storage if digestion isn’t ready).After
removing the supernatant from the last washing, add approximately 500uL of 100mM NH4OAc (pH 7.4) with
0.05% (w/w) NaN3. Store at 4˚C.
16.Mix 1mg of substrate/protein to 300uL of phosphate
buffer (pH 7.4).
17.Add substrate solution to pronase beads.
18.Place samples in the incubator with gentle agitation using
these conditions:
a.Temperature –37˚C, Speed- 10, Time –Allow digestion
for pre-determined time (3-48hrs) depending on peptide length expected.
19.Centrifuge the suspension using a microfuge (~1
minute), and recover the supernatant in a new microfuge tube. Use great care to avoid transferring any beads along with the glycopeptides supernatant.
20.Continue with SPE clean up using the following
conditions:
a.Condition –9mL H2O, 9mL 80% ACN in 0.1% TFA,
15mL H2O
b.Load Sample
c.Desalt with 9mL H2O
d.Elute with 9mL 40% ACN in 0.05% TFA
21.Concentrate/Dry using a centrifugal evaporator.
22.Analysis
a.If using 1mg of standard protein, sample can be
analyzed using Chip-TOF/Q-TOF after a 5x dilution.
Injection volume should be 2-3µL.
**NOTE 1: Do not use buffers or reagents that introduce free amine groups (e.g. Tris buffers) until after the ethanolamine blocking step to prevent interference with successful enzyme immobilization.**NOTE 2: The coupling efficiency and the stability of enzyme coupled beads are pH sensitive. Always ensure that all solutions are buffered at the appropriate pH.。