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Protocols

Millipore ZipTip (For Reversed-Phase)

Materials

ZipTip_C18 tips- a standard bed of 0.6 uL for sample elution in 1 to 4 uL. Appropriate for peptides and low molecular weight proteins.

ZipTip_C4 tips- a micro bed of 0.2 uL for sample elution in <1 uL. Appropriate for low to intermediate molecular weight proteins, proteins over 100,000 Da.

The procedure also requires a compatible 10 uL pipettor. For simultaneous processing of multiple samples, Millipore recommends the Biohit Proline™ Multi-channel Pipettor.

Solutions (make fresh daily):	ZipTipC18 tips	ZipTipC4 tips
Wetting solution	100% Acetonitrile (ACN)	100% Acetonitrile (ACN)
Sample Prep	0.5% TFA in ddH2O	0.5% TFA in ddH2O(optional) Guanidine-HCl 1-6 M maybe added
Equilibration solution	0.1% TFA in ddH2O	0.1% TFA in ddH2O
Wash solution	0.1% TFA in ddH2O	0.1% TFA in ddH2O
Elution solution	0.1% TFA/ 50% ACN With or without Matrix	0.1% TFA/ 50%ACN With or without Matrix

Procedure

ZipTipC₁₈ tips

Maximum binding occurs in the presence of TFA (0.1%) or other ion-pairing agents. To maximize analyte binding, use the appropriate sample preparation solution. Ensure that the final solution has a pH < 4.

ZipTip_C4 tips

Optimal binding may require a chaotropic agent (guanindine-HCl at a final concentration of 1-6 M). If the sample does not already contain chaotropic salts, add them a few minutes prior to binding. These salts will be removed during the wash step following binding.

Equilibrate the ZipTip pipette tip for sample binding

1. Depress pipettor plunger to a dead stop. Using the maximum volume setting of 10 uL, aspirate wetting solution into the tip. Dispense to waste. Repeat 2x.
2. Aspirate equilibration solution. Dispense to waste. Repeat 2x.

Bind and wash the peptides or proteins

Follow these steps after equilibrating the ZipTip pipette tip:

1. Bind peptides and/ or proteins to ZipTip pipette tip by fully depressing the pipette plunger to a dead stop. Aspirate and dispense the sample 7-10 cycles for maximum binding of complex mixtures.
2. Aspirate wash solution into tip and dispense to waste. Repeat 4x.

NOTE: 5% methanol in 0.1% TFA/ water wash can improve desalting efficiency. Additional washing may be required for electrospray MS.

Elute the peptides or proteins

For ZipTipC₁₈ (standard bed format) and ZipTip_C4, pipette tips, dispense 1-4 uL of elution solution into a clean vial using a standard pipette tip. In the case of ZipTipu-C₁₈ (micro bed format) pipette tips, dispense 0.5-2 uL of elution solution into a clean vial.

Caution: Acetonitrile and methanol are volatile and evaporation can occur rapidly. If this occurs, add more eluant to recover sample.

Carefully, aspirate and dispense eluant through ZipTip pipette tip at least three times without introducing air into the sample. Sample recovery can be improved (at the expense of concentration) by increasing the volume to 5 uL.

For direct spotting onto a MALDI-TOF MS target

Elute with or without matrix elution solution.

1. Pipette 0.5-4 uL of desalted-concentrated sample directly onto target by depressing plunger until appropriate volume is dispensed.
2. Save or discard the remaining solution.

In-Gel Digest Procedure

• Obtain an image of the gel from which the bands are excised, this is important for determining the volume to inject into the mass spectrometer.
• Wearing gloves and lab coat, wipe down ALL surfaces in the hood with methanol/water moistened lint-free cloth. Try to keep contaminants to a minimum such as keratin.
• Use only polypropylene low binding tubes and pipette tips for this procedure.
• Wipe razor blades with methanol-soaked lint-free cloth.

Prepare the following solutions:

These are Final concentrations; I suggest that you prepare a 50 mM stock of ABC to start with.

25 mM NH₄HCO₃ (100 mg/50 mL) (ABC)
25 mM NH₄HCO₃ in 50% ACN
10mM dithiothreitol (DTT) in 25 mM NH₄HCO₃ (1.5mg/mL)
55mM iodoacetamide (IA) in 25mM NH₄HCO₃ (10mg/mL)
15 ng/mL trypsin in 25 mM NH₄HCO₃ (freshly diluted)

1. >Dice each gel slice into small pieces (1 .2 mm) and place into 0.65 mL tubes.
2. Add 50µL (or enough to cover) of 25mM ABC to gel plugs and hold at room temperature for 10 min.
3. Purge liquid by using gel loading pipette tip, add 50 µL ACN and hold at room temperature for 10 minutes.
4. Repeat steps 2 and 3 once or twice.
5. Speed Vac the gel pieces to complete dryness (~10 min.)

For low-level proteins (<1 pmol), especially those separated by 1-D SDS-PAGE, reduction and alkylation is recommended. These procedures are performed after step 5.

1. Add 30 µL 10 mM DTT in 25 mM NH4CO3. Vortex, spin briefly. Allow reaction to proceed at 56°C for 45 minutes.
2. Allow gel plugs to cool for 20 minutes.
3. Remove supernatant and add 30 µL of 55 mM iodoacetamide to the gel pieces. Prepare the iodoacetamide in the dark immediately before using. Vortex and spin briefly. Allow reaction to proceed in the dark for 45 minutes at room temperature.
4. Purge liquid and repeat step 2 & 3 twice.
5. Add 10 µL or just barely cover the gel pieces of the trypsin solution to rehydrate the gel plugs, leave on ice for 10 minutes. This volume will vary from sample to sample, but on average ~5-25uL is sufficient.
6. Add 15 µL ABC as needed to cover the gel pieces and incubate at 37°C for 4 hours-overnight.
7. Add 7 µL of 1% (final) formic acid to quench the reaction. Amount may vary based on your final digestion volume.
8. All volumes can be adjusted, however, be sure to calculate for final concentrations.

Extraction of Peptides

1. Transfer the digest solution (aqueous extraction) into a clean 0.65mL siliconized tube.
2. To the gel pieces, add 30uL (enough to cover) of 50% ACN/5% formic acid, vortex 20-30 min, spin, sonnicate 5 min. Repeat.
3. Vortex the extracted digest, spin and Speed Vac to reduce volume to 10uL.
4. Either proceed with cleanup ZipTip (Millipore) or analyze with LC-MS. Add formic acid to a final concentration of 5%.
5. When analyzing low levels of protein, concentrate the peptides by utilizing ZipTips.

Sample is now ready for mass spectrometric analysis.