TITLE: BAF_Protocol_003 Desalting
AUTHORS: nesf

[DESCRIPTION]
The procedure is used to desalt and purify peptides from solution or in-gel digestion. This step results in minimal losses but increases analytical column life on the mass spectrometer. At the end of the procedure salts and small molecules should be washed away from peptides. 

[BEFORE_START]
Amount of total peptide to choose between C18 10 µL or 100 10 µL tips:

10 µL tip can bind up to 8 µg of total peptide.

100 µL tip can bind up to 80 µg of total peptide.


Material Preparation

• Acidic solution: 2.5% FA
• Wetting solution: 70:30 ACN:water; 20 μL or 200 μL per sample
• Equilibration solution: 0.1% FA in water; 20 μL or 200 μL per sample
• Rinse solution: 0.1% FA in 5% ACN:water; 20 μL or 200 μL per sample
• Elution solution: 0.1% FA in 70% ACN:water for ESI-MS, up to 100 μL per sample

[GUIDELINES]
Note 1: For optimal flow and peptide recovery, do not introduce air through the tip at any time during the procedure.

Note 2: This protocol is for C18 tips from Thermo Scientific. Pierce™ C18 Spin Tips & Columns part number for 10  μL tips 87782 and part number for 100  μL tips 87782. 

[STEPS]
SECTION: Procedure for the 10 μL C18 Tips
1. Set the micropipette to 10 μL and secure the pipette tip tightly to the end for optimum tip-to-micropipette seal and sample aspiration.

SECTION: Procedure for the 10 μL C18 Tips
2.  Resuspend the sample with 0.1% FA (formic acid) or adjust sample to 0.1-1.0% FA using 2.5% FA to 12 µL total volume. Check that pH ~3.0. Needs to be pH 3 or slightly lower for efficient binding.

SECTION: Procedure for the 10 μL C18 Tips
3.  Wet tip by aspirating 10 μL of 70% ACN (acetonitrile) in water and then discarding solvent. Repeat 5 times.

SECTION: Procedure for the 10 μL C18 Tips
4.  Equilibrate tip  by aspirating 10 μL of 0.1% FA and discarding solvent. Repeat 5 times.

SECTION: Procedure for the 10 μL C18 Tips
5. Aspirate up to 10 μL of sample (prepared in Step 2) onto the C18 tip. For the maximum efficiency, dispense and aspirate the sample for 30 cycles (in the sample tube - slowly).

SECTION: Procedure for the 10 μL C18 Tips
6.  Rinse the tip by aspirating 10 μL of 0.1% FA with 5% ACN and discarding solvent. Washing out salts and other small molecules that do not stick to C18. Repeat 5 times.

SECTION: Procedure for the 10 μL C18 Tips
7.  Elute the sample: Slowly aspirate 5-10 μL of 0.1% formic acid in 70% ACN and dispense into a pre-cleaned 0.6 mL tube. Dispense and aspirate in the tube 30 cycles.

SECTION: Procedure for the 10 μL C18 Tips
8.  Rinse the tip by aspirating the 10 μL of 0.1% FA with 5% ACN and discarding solvent. Repeat 5 times. Same as step 4.

SECTION: Procedure for the 10 μL C18 Tips
9. Repeat steps 5-7. Second elution will be in same tube as first elution.

SECTION: Procedure for the 100 μL C18 Tips
11. For larger tip (higher capacity) change volumes to 100uL with elution using 50-100uL. Final reconstitution volume will also be adjusted. 

SECTION: Procedure for the 100 μL C18 Tips
15. Dry the eluted sample in speed vac. Keep dry at -80C until MS analysis or directly resuspend in 20-40 µL of 0.1% formic acid, 30 sec vortex, centrifuge at 9,000xg for 5 min. Pipette 10 µL of sample not touching bottom) into autosampler vial and keep the backup sample at -20C.
SECTION: Procedure for the 10 μL C18 Tips
15. 
SECTION: Procedure for the 10 μL C18 Tips
10. Dry eluted sample in a speedvac and store at -80C until MS analysis. If going directly to MS analysis, then add 20-40uL 0.1% FA to tube, vortex, microfuge for 15 minutes at max speed, and take off 10uL into autosampler vial (do not touch bottom of tube).