Phosphopeptide Enrichment

IMAC One-Step Global Phosphopeptide Enrichment

1. Start with ~1mg normally processed lyophilized peptides, it is also possible to start with desalted peptides still in 50% acetonitrile if they are concentrated enough (>6 mg/ml.)
2. Materials needed
   1. Ni-NTA Superflow beads
   2. 500 mM potassium phosphate pH 7 (prepared from mixing 1M K₂HPO₄ and 1M KH₂PO₄)      
   3. 100 mM FeCl₃, freshly prepared
   4. 100 mM EDTA pH 8
   5. 80% ACN, 0.1% TFA
   6. 5% formic acid in water
   7. 50% ACN, 0.25% formic acid
   8. Desalting Vacuum Manifold
   9. NEST desalting tips (ultraMicroSpin Column, Silica C18)
   10. 2 mL blank biospin columns (Pierce)
3. Place and Wet C18 Desalting tips
   1. Place desalting tip on vacuum manifold: Cut 0.5 cm off of desalting tip and place in stopcock fitting.  Twist and press tip lightly to form an airtight seal. Turn on vacuum to 5~PSI 
   2. Wet tip with 150 µL 80% ACN, 0.1% TFA. Leave ~10 µL to avoid drying out.
4. Prepare Fe-NTA beads from Ni-NTA beads. So far I am only using beads and FeCl₃ I prepared fresh each time. Beads are sticky so use or make wide bore 1 ml and 200 µL tips.  Current protocol takes place with desalting vacuum manifold.
   1. An empty 2 mL spin/vac column is used to make Fe-NTA beads. Prepare and place column on top of manifold.
   2. Transfer 200 µL of beads (~400 µL slurry) to vacuum column.
   3. Add ~500 µL 100 mM EDTA, pipette to mix 6-8 times, incubate 30 seconds, remove liquid to wash.
   4. Repeat three times to fully strip away Nickel. Beads should lose blue color and be colorless.
   5. Wash beads with ~500 µL H2O, repeat once
   6. Add ~500 µL 100 mM FeCl₃, pipette to mix 6-8 times, incubate 1 minute, remove liquid to wash.
   7. Repeat three times to incorporate Fe. Beads should acquire orange tint due to Fe.
   8. Add ~500 µL H20, pipette to mix 6-8 times, remove liquid to wash.
   9. Repeat 2 times to remove excess Fe. Beads should have orange tint but liquid above be colorless.
   10. Rinse beads with ~500 µL of 0.5% FA (I think this gets rid of excess Fe).
   11. Add ~600 µL H2O to beads to make a 25% slurry.
   12. While mixing, pipette ~60 µL slurry from vac column to each labeled C18 microspin column/tip.
   13. This should give 10-15 µL beads in each column. This corresponds to width of a Sharpie marker.
5. Prepare, Add and incubate Peptide Sample -> to ~75% ACN 0.15 % TFA final concentration before loading
   1. Dissolve ~1mg of peptides in 100 uL 50/50 ACN/HPLCwater. Vortex well, make sure well dissolved
   2. Add 100 µL of 100% ACN, mix well.
   3. Add 3 µL of 10% TFA, mix well.
   4. Spin down any insoluble peptides
6. Add peptides in solution to Fe-NTA beads in to corresponding, labeled desalting tip.
7. Incubate 1-2 minutes,
8. Pipette 8-10x to mix, incubate 1-2 more min.
9. Wash, Elute and Desalt Phosphopeptides
10. After incubating drain liquid,
11. Add 200 µL 80% ACN, 0.1% TFA, pipette to mix 8-10 times, remove liquid to wash. Repeat 2 times.
12. Rinse 4th time, but DO NOT pipette up and down and try to rinse any remaining liquid down.
13. Add 200 µL 0.5% FA, no need to mix, drain liquid to wash.
14. Repeat once, this equilibrates C18 column.
15. Add 200 µL 500 mM phosphate pH7, pipette to mix 8 times, incubate 2-3 minutes, drain liquid.
16. Repeat once, this elutes phosphopeptides from beads onto C18 column.
17. Add 200 µL 0.5% FA, no need to mix, incubate 15 seconds, drain liquid to wash.
18. Repeat twice to remove phosphate from C18 column.
19. Take columns off of vacuum block and elute by spinning instead. place column on labeled sample tube with 200 µL tip adaptor. Sample tube can be placed in 2 ml as a holder, thus turning it into spin column
20. Add 60 µL 50 % ACN 0.25% FA, spin into collection tube.
21. Repeat once to finish eluting off of C18 column.
22. Freeze eluate on dry ice and dry on speed vac or lyophilizer.
23. Ready for MS, resuspend in 25-50 µL 0.1% F.A. or loading buffer when sample will be run on MS.