Protocols

The sample amount needed for IRMS analyses is a function of how much of the element of interest is present in the sample. Please contact Dr. Mihai Lefticariu before weighing or submitting samples. Please note the Mass Spectrometry Facility does not run samples that are damaged, insufficiently prepared, or are considered to pose a hazard for the instruments if run.

1. δ¹³C, δ¹⁵N, δ²H, δ¹⁸O and weight %C, N, H, and O in solid organic and inorganic samples with natural isotope abundances by CF-EA-IRMS and CF-TCEA-IRMS

   Please make sure the samples are ground/cut as fine as possible and thoroughly dried before weighing and packing them into tin (for EA) or silver (for TCEA) capsules. The capsules containing the sample should be gently crushed into round shapes without protruding ‘spikes’ or ‘winglets’ in order to optimize their drop into the combustion tube during runs. Samples requiring demineralization as part of preparation such as soil, sediment, and coal, for example, should be thoroughly washed with deionized water and dried before packing them into capsules to remove any traces of acid.

2. δ¹³C, δ²H, weight %H and weight %C in oil samples with natural isotope abundances by CF-EA-IRMS and CF-TCEA-IRMS

   Use either a capsule designed for liquid samples or make sure that the regular capsule does not leak nor has oil on the outside.

3. δ²H and δ¹⁸O in water samples with natural isotope abundances by CF-TCEA Liquid Injection-IRMS

   Natural water samples should be filtered to remove all suspended solids and the pH measured prior to sending them to the facility. A minimum sample volume of 5 ml is recommended.

4. δ¹⁸O in water samples with natural isotope abundances by Gas Bench-Water equilibration-IRMS

   A minimum sample volume of 10 ml is recommended.

5. δ¹³C  and δ¹⁸O in carbonate samples with natural isotope abundances by off-line He flush/fill and acidification and Gas Bench-IRMS

  The mineralogic composition of the sample should be known.

Below you will find a number of guidelines that should be followed when preparing a sample for mass spectrometric analysis.

• All samples should be submitted in polypropylene microcentrifuge tubes/vials (for peptides or proteins samples) or deactivated glass vials with Teflon-lined screw caps (for small molecule samples). Use a small volume insert if a limited amount of sample is available.

• All sample tubes must be labeled with a sample ID and be accompanied by a sample submission form.

• For MALDI analysis, please submit at least 1mg of purified solid sample or 50uL of (at least) 10pmol/mL sample solution prepared in compatible solvent(s) or a volatile buffer.

• For electrospray analysis, please submit 50uL of (at least) 10pmol/mL sample solution prepared in 50% acetonitrile.

• Solvents that are compatible with MALDI include acetonitrile, methanol, isopropanol, ethanol and water.

• A volatile buffer such as ammonium acetate should be used whenever pH control is necessary. The maximum concentration that can be tolerated is 50 mM.

• Allowable detergents include n-octyl-glucoside, n-dodecyl-glucoside, octanoyl-N-methylglucamide and decanoyl-n-methylglucamide

• Avoid using:

  • Tris, phosphates, citrates, and HEPES

  • Urea and guanidinium salts

  • TFA (acts as strong ion-pairing agent in electrospray)

  • Detergents such as SDS, CHAPS, PEG, Tween, and Triton

  • DMSO, acetates and  glycerol

  The presence of any of the above chemicals can completely suppress ionization of the sample making the difference between a successful or failed analysis.

1. Remove the gel from the electrophoresis chamber and place enough 0.5% Coomassie Blue G-250 (prepared in 50% methanol/ 10% acetic acid) to cover the gel. Use freshly washed labware that has never been in contact with nonfat milk, BSA or any other protein blocking agent to prevent carryover contamination. Stain for about 5 minutes.
2. Discard stain and rinse briefly with MilliQ water to remove most of the residual stain in the glassware.
3. Destain with 40% HPLC grade methanol/ 10% acetic acid, replacing the solution every 10-20 minutes until faint bands are observed. Kimwipes rolled up into balls can be added to speed up the destaining.
4. Continue destaining with MilliQ water until bands are very clean. Usually we destain overnight in MilliQ water with several Kimwipes present. Bands/spots can now be excised and submitted for analysis.

Modified from “Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels” A. Shevchenko, M. Wilm, O. Vorm, M. Mann Analytical Chemistry 1996, 68 (850 – 858).

Convenient Recipes

1. Sensitization Solution (0.02% sodium thiosulfate) – 0.1 g sodium thiosulfate in 500 mL MilliQ water
2. Staining Solution (0.1% silver nitrate) - 0.5 g silver nitrate in 500 mL MilliQ water (use glass container only!)
3. Developing Solution (0.04% formalin in 2% sodium carbonate) - 125 μL formalin (37% formaldehyde solution) and 15 g sodium carbonate in 500 mL MilliQ water

Staining Procedure

1. Immediately following electrophoresis, remove gel from plates and notch the upper right corner. Place gel in the fixing solution (50% methanol: 12% glacial acetic acid) and gently shake overnight.
2. Discard the fixing solution. Wash the gel with 50% methanol for 20 minutes.
3. Discard the wash solution. Wash the gel with MilliQ water for 20 minutes.
4. Repeat wash in step IIc.
5. Discard wash solution. Place gel in sensitization solution (0.02% sodium thiosulfate) for 1 minute.
6. Discard sensitization solution. Wash the gel with MilliQ water for 1 minute.
7. Discard wash solution. Wash the gel a second time in MilliQ water for 1 minute.
8. Discard the wash solution. Place the gel in the staining solution (0.1% silver nitrate, chilled) for 20 minutes.
9. Discard the staining solution. Wash the gel with MilliQ water for 1 minute.
10. Discard the wash solution. Wash the gel a second time with MilliQ water for 1 minute.
11. Discard the wash solution. Place the gel in the developing solution (0.04% formalin in 2% sodium carbonate). Time is variable depending on desired staining intensity.
12. Discard developing solution. Place gel in stopping solution (5% glacial acetic acid) for 10 minutes. m. Discard stopping solution. Store gel in 1% glacial acetic acid at 4°C.

Notes:

• Wear clean non-latex gloves at all times to avoid keratin contamination.
• Wash Pyrex dish(es) with detergent and wash with copious volumes of deionized water prior to starting silver staining protocol.

Material Preparation

1. Trypsin Stock: Prepare the Trypsin Stock solution in a concentration of 0.1ug/uL by adding 200uL of Trypsin resuspension buffer to 20ug of Trypsin. Store this solution at -20°C for up to two months.
2. Digestion Buffer: Weigh 10 mg of the Ammonium Bicarbonate and dissolve in 2.5mL of ultrapure water for a final concentration of ~50mM. This solution can be stored at 4°C for up to two months.
3. Reducing Buffer: Weigh 8 mg of DDT and dissolve with 500uL of ultrapure water for a final concentration of ~100mM. Store reducing buffer at -20°C.
4. Alkylation Buffer: Prepare Alkylation Buffer just before use. Weigh 9 mg of Iodoaceamide and add it to a foil-wrapped tube to avoid exposure to light. Add 500uL of ultrapure water for a final concentration of ~100mM. Do not store excess.
5. Protein Concentrations: The procedure described below has been found to work well with protein concentrations in the range of 0.1 – 1.0 mg/mL. This concentration range will allow you to work with from 1.0ug to 10ug of protein, respectively, in 10uL total volume for the digestions.

Procedure for In-Solution Digestion

1. Reduction and Alkylation
   1. Add 15uL of Digestion Buffer and 1.5uL of Reducing Buffer to a 0.5mL microcentrifuge tube.
   2. Add 10uL of the protein solution to the tube and adjust the final volume to 27uL with ultrapure water.
   3. Incubate sample at 95°C for 5 minutes. Allow sample to cool.
   4. Prepare Alkylation Buffer as described in the Material Preparation Section. Add 3uL of Alkylation Buffer to the tube and incubate in the dark at room temperature for 20 minutes.
2. Digestion
   1. Prepare Trypsin as described in the Material Preparation Section.
   2. Add 1uL Trypsin to reaction tube and incubate at 37°C for 3 hours.
   3. Add an additional 1uL Trypsin and incubate the reaction at 30°C overnight.
   4. Spot 0.5 µL of α-cyano-4-hydroxycinnamic acid matrix (10 mg/mL in 50% HPLC grade acetonitrile, 0.1 % TFA) on MALDI sample plate followed by 0.5 µL of sample. Unused sample may be frozen at -20°C in case further analyses are required.
   5. Allow spots to dry completely and perform MALDI MS data collection as soon as possible.

Notes

• Always use non-latex gloves when handling samples, keratin and latex proteins are potential sources of contamination.
• Never re-use any solutions, abundant proteins will partially leach out and contaminate subsequent samples.

References

1. Procedure modified from Pierce In-Solution Tryptic Digestion Kit.

Washing/Destaining

1. The SDS-PAGE gel should only be stained with high quality Coomassie Brilliant-Blue G-250. Use only MilliQ water for destaining and reagent preparation.
2. Wash 1.5 mL Eppendorf microcentrifuge tubes with 50% HPLC grade acetonitrile: MilliQ water.
3. Excise gel band(s), cut into 1mm pieces and place into a washed tube(s).
4. Equilibrate gel pieces with 500 µL 100 mM ammonium bicarbonate (aq) buffer for 20 minutes at room temperature with gentle agitation.
5. Wash gel pieces with 500 µL wash solution (50 mM ammonium bicarbonate, 50% acetonitrile) until Coomassie is removed (3 - 4 x 15 min, with gentle agitation).
6. Rinse gel pieces briefly with 500 µL 100% HPLC grade acetonitrile. Discard acetonitrile, and allow gel pieces to air-dry.

Reduction/Alkylation

1. Reduce the in-gel protein with dithiothreitol (150 µL 10mM DTT/ 100mM ammonium bicarbonate) for 30 minutes at 56° C.
2. Cool the sample to room temperature, remove and discard DTT solution.
3. Alkylate the in-gel protein with iodoacetamide (100 µL 50 mM IAA/100 mM ammonium bicarbonate) in the dark at room temperature for 30 minutes. Remove and discard IAA solution.
4. Wash sample at room temperature for 15 minutes with 500 µL of wash solution.
5. Rinse gel pieces briefly with 500 µL 100% HPLC grade acetonitrile. Discard acetonitrile and allow to air-dry.

Digestion/Extraction

1. Rehydrate gel pieces for 60 minutes at 4°C (on ice) in 30 µL of 0.02 µg/µL trypsin (Promega, modified porcine, TPCK-treated, Cat#: V5111) in 50mM ammonium bicarbonate. (Be sure that enough volume is added to ensure complete rehydration of gel pieces)
2. Remove trypsin solution and replace with just enough 50mM ammonium bicarbonate to cover gel pieces. Incubate at 37°C for 16-18 hours (overnight).
3. Add 7 μL of 1% formic acid to stop the action of trypsin. Spin down the sample and transfer supernatant (containing tryptic peptides) to a clean 0.5 mL Eppendorf microcentrifuge tube and keep supernatant on ice.
4. Rinse the gel pieces with a small volume of 0.1% formic acid and transfer solution to tube from step IIIc.
5. Store the samples at -20°C until ready for analysis.

Notes

• Always use non-latex gloves when handling samples, keratin and latex proteins are potential sources of contamination.
• Never re-use any solutions, abundant proteins will partially leach out and contaminate subsequent samples.

References

1. Havlis J, Thomas H, Sebela M, Shevchenko A: Fast-response proteomics by accelerated in-gel digestion of proteins. Analytical Chemistry 75: 1300-1306 (2003).
2. Hellman U, Wernstedt C, Góñez J, Heldin C-H: Improvement of an "in-gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Analytical Biochemistry 224: 451-455 (1995).
3. Jiménez CR, Huang L, Qiu Y, Burlingame AL: In-gel digestion of proteins for MALDI-MS fingerprint mapping. In: Coligan JE (ed) Current Protocols in Protein Science, pp. 16.4.1-16.4.5. John Wiley & Sons, Inc., Brooklyn, N.Y. (1998).
4. Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P: In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Analytical Biochemistry 203: 173-179 (1992).
5. Shevchenko A, Wilm M, Vorm O, Mann M: Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Analytical Chemistry 68: 850-858 (1996).

Washing/Destaining

1. The SDS-PAGE gel should only be stained with high quality Coomassie Brilliant Blue G-250. Use only MilliQ water for destaining and reagent preparation.
2. Once gel is destained and background is minimal, excise stained spots and transfer into a clean 1.5 mL Eppendorf microcentrifuge tube.
3. Wash gel with 500 µL of wash solution (50% HPLC grade acetonitrile, 50mM ammonium bicarbonate) and incubate at room temperature for 15 min with gentle agitation (vortex mixer or shaker on lowest setting). Remove solution with a pipette and discard.
4. Wash gel two more times with 500 µL of wash solution (15 min each) or until the Coomassie dye has been completely removed (usually 4 washes with G-250 coomassie).
5. Dehydrate gel in 500 µL 100% HPLC grade acetonitrile briefly. Remove with pipette and discard, taking care not to pull gel piece(s) into pipette tip.
6. Completely air-dry each gel piece with the cap just slightly open. Care should be observed when handling the tube once the gel is dry because it will “jump” out due to static electricity.

Digestion/Extraction

1. While gel is drying prepare protease digestion solution. [Typically, this is modified sequencing grade trypsin (Product number V5111, Promega, Madison, WI)]. Resuspend lyophilized trypsin (20 µg/vial) in 20 µL of the 50mM acetic acid solution provided with trypsin, yielding a 1 µg/µL stock solution. Dilute that stock to 1 µg/50 µL with 50mM ammonium bicarbonate (50 fold dilution), keeping in mind the number of gel pieces you have to digest. Store the remaining trypsin stock at –70° C. Do not freeze-thaw trypsin stock solutions more than once.
2. Rehydrate the gel with a minimal volume of trypsin protease digestion solution. Use 20 µL for small gel pieces, 30-40 µL for larger gel pieces. Add more if gel pieces absorb all the liquid.
3. Incubate at 4°C (on ice) for 1 hour. At this point most of the trypsin will be absorbed into the gel pieces.
4. Remove trypsin solution and replace with just enough 50mM ammonium bicarbonate to cover gel pieces. Incubate at 37°C for 16-18 hours (overnight).
5. Add 7 μL of 1% formic acid to stop the action of trypsin. Spin down the sample and transfer supernatant (containing tryptic peptides) to a clean 0.5 mL Eppendorf microcentrifuge tube and keep supernatant on ice.
6. Rinse the gel pieces with a small volume of 0.1% formic acid and transfer solution to tube from step IIIe.
7. Store the samples at -20°C until ready for analysis.

Notes

• This protocol does not contain a reduction and alkylation step, and assumes these steps have been performed during IPG strip equilibration, prior to running the second dimension.
• Always use non-latex gloves when handling samples, keratin and latex proteins are potential sources of contamination.
• Never re-use any solutions, abundant proteins will partially leach out and contaminate subsequent samples.

References

1. Havlis J, Thomas H, Sebela M, Shevchenko A: Fast-response proteomics by accelerated in-gel digestion of proteins. Analytical Chemistry 75: 1300-1306 (2003).
2. Jiménez CR, Huang L, Qiu Y, Burlingame AL: In-gel digestion of proteins for MALDI-MS fingerprint mapping. In: Coligan JE (ed) Current Protocols in Protein Science, pp. 16.4.1-16.4.5. John Wiley & Sons, Inc., Brooklyn, N.Y. (1998).

Washing/Destaining

1. Wash 0.5 mL Eppendorf microcentrifuge tubes with 50% HPLC grade acetonitrile, 0.1% trifluoroacetic acid (TFA).
2. Using a freshly stained gel, cut the protein bands at the margin of the visible bands using a new razor blade. Wash the razor blade after cutting each band. Cut gel band into 1mm cubes and place gel pieces in pre-washed 0.5 mL microcentrifuge tube.
3. Add 200 μL MilliQ water and shake for 1 hour to remove the acetic acid. Remove and discard supernatant.
4. Destain by adding 25 μl of 15mM potassium ferricyanide and 25 μL of 50mM sodium thiosulfate. Shake gently until the band pieces go clear (i.e. until all the silver is removed). Remove and discard the supernatant. The gel pieces will now appear yellow in color.
5. Add 200 μL of 200mM ammonium bicarbonate, pH 8. Shake for 15 minutes. Gel pieces should be colorless. Remove and discard supernatant.
6. Add 200 μL of 50:50 (v/v) 200 mM ammonium bicarbonate: acetonitrile. Shake gently for 15 minutes. Remove and discard supernatant.
7. Dehydrate gel piece by adding 200 μL HPLC grade acetonitrile. Remove immediately and discard supernatant. Allow residual acetonitrile to evaporate completely.

Reduction/Alkylation

1. Reduce the in-gel protein with dithiothreitol (150 μL 10mM DTT in 100mM ammonium bicarbonate) for 30 minutes at 56°C.
2. Allow sample to cool to room temperature, remove and discard DTT solution.
3. Alkylate the in-gel protein with iodoacetamide (100 μL 50mM IAA in 100mM ammonium bicarbonate) in the dark at room temperature for 30 minutes. Remove and discard IAA solution.
4. Add 200 μL of 50:50 (v/v) 200 mM ammonium bicarbonate: acetonitrile. Shake gently for 15 minutes. Remove and discard supernatant.
5. Dehydrate gel piece by adding 200 μL HPLC grade acetonitrile. Remove immediately and discard supernatant. Allow residual acetonitrile to evaporate completely.

Digestion/Extraction

1. Rehydrate each gel piece at 4°C in 10-20 μL 0.02 μg/μL of Promega Sequencing Grade Modified Trypsin prepared in 50mM ammonium bicarbonate. If additional solvent is required to cover gel piece, use 50 mM ammonium bicarbonate (NO Trypsin).
2. Close the microcentrifuge tubes tightly and incubate overnight at 37°C.
3. Add 7 μL of 1% formic acid to stop the action of trypsin. Spin down the sample and transfer supernatant (containing tryptic peptides) to a clean 0.5 mL Eppendorf microcentrifuge tube and keep supernatant on ice.
4. Rinse the gel pieces with a small volume of 0.1% formic acid and transfer solution to tube from step IIIc.
5. Store the samples at -20°C until ready for analysis.

Notes

• Digestion protocol may be conveniently stopped when the water is added before shaking (step Ic). Freeze the bands/spots solution at -20°C. When ready to continue digestion, thaw bands/spots and shake for 1 hour.
• Always use non-latex gloves when handling samples, keratin and latex proteins are potential sources of contamination.
• Never re-use any solutions, abundant proteins will partially leach out and contaminate subsequent samples.

References

1. Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM: Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity. Electrophoresis 20: 601-605 (1999).
2. Havlis J, Thomas H, Sebela M, Shevchenko A: Fast-response proteomics by accelerated in-gel digestion of proteins. Analytical Chemistry 75: 1300-1306 (2003).
3. Hellman U, Wernstedt C, Góñez J, Heldin C-H: Improvement of an "in-gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Analytical Biochemistry 224: 451-455 (1995).
4. Jiménez CR, Huang L, Qiu Y, Burlingame AL: In-gel digestion of proteins for MALDI-MS fingerprint mapping. In: Coligan JE (ed) Current Protocols in Protein Science, pp. 16.4.1-16.4.5. John Wiley & Sons, Inc., Brooklyn, N.Y. (1998).
5. Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P: In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Analytical Biochemistry 203: 173-179 (1992).
6. Shevchenko A, Wilm M, Vorm O, Mann M: Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Analytical Chemistry 68: 850-858 (1996).

Washing/Destaining

1. Wash 0.5 mL Eppendorf microcentrifuge tubes with 50% HPLC grade acetonitrile, 0.1% trifluoroacetic acid (TFA).
2. Using a freshly stained gel, cut the protein spots at the margin of the visible bands using a new disposable transfer pipette. Discard the pipette after cutting each spot. Place gel spots in pre-washed 0.5 mL microcentrifuge tube.
3. Add 200 μL MilliQ water and shake for 1 hour to remove the acetic acid. Remove and discard supernatant.
4. Destain by adding 25 μl of 15mM potassium ferricyanide and 25 μL of 50mM sodium thiosulfate. Wait for 5 - 10 min until the band pieces go clear (i.e. until all the silver is removed). Remove and discard the supernatant. The gel pieces will now appear yellow in color.
5. Add 200 μL of 200mM ammonium bicarbonate, pH 8. Shake for 15 minutes. Gel pieces should be colorless. Remove and discard supernatant.
6. Add 200 μL of 50:50 (v/v) 200 mM ammonium bicarbonate: acetonitrile. Shake gently for 15 minutes. Remove and discard supernatant.
7. Dehydrate gel piece by adding 200 μL HPLC grade acetonitrile. Remove immediately and discard supernatant. Allow residual acetonitrile to evaporate completely.

Digestion/Extraction

1. Rehydrate each gel piece at 4°C in 10-20 μL 0.02 μg/μL of Promega Sequencing Grade Modified Trypsin prepared in 50mM ammonium bicarbonate. If additional solvent is required to cover gel piece, used 50 mM ammonium bicarbonate (NO Trypsin).
2. Close the microcentrifuge tubes tightly and incubate overnight at 37°C.
3. Add 7 μL of 1% formic acid to stop the action of trypsin. Spin down the sample and transfer supernatant (containing tryptic peptides) to a clean 0.5 mL Eppendorf microcentrifuge tube and keep supernatant on ice.
4. Rinse the gel pieces with a small volume of 0.1% formic acid and transfer solution to tube from step IIIc.
5. Store the samples at -20°C until ready for analysis.

Notes

• This protocol does not contain a reduction and alkylation step, and assumes these steps have been performed during IPG strip equilibration, prior to running the second dimension.
• Digestion protocol may be conveniently stopped when the water is added before shaking (step Ic). Freeze the spots solution at -20°C. When ready to continue digestion, thaw bands/spots and shake for 1 hour.
• Always use non-latex gloves when handling samples, keratin and latex proteins are potential sources of contamination.
• Never re-use any solutions, abundant proteins will partially leach out and contaminate subsequent samples.

References

1. Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM: Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity. Electrophoresis 20: 601-605 (1999).
2. Havlis J, Thomas H, Sebela M, Shevchenko A: Fast-response proteomics by accelerated in-gel digestion of proteins. Analytical Chemistry 75: 1300-1306 (2003).
3. Hellman U, Wernstedt C, Góñez J, Heldin C-H: Improvement of an "in-gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Analytical Biochemistry 224: 451-455 (1995).
4. Jiménez CR, Huang L, Qiu Y, Burlingame AL: In-gel digestion of proteins for MALDI-MS fingerprint mapping. In: Coligan JE (ed) Current Protocols in Protein Science, pp. 16.4.1-16.4.5. John Wiley & Sons, Inc., Brooklyn, N.Y. (1998).
5. Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P: In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Analytical Biochemistry 203: 173-179 (1992).
6. Shevchenko A, Wilm M, Vorm O, Mann M: Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Analytical Chemistry 68: 850-858 (1996).

Materials

• Wetting solution (100% HPLC-grade acetonitrile)
• Stock solution (1% TFA in MilliQ water): 990 μL MilliQ water, 10 µL TFA
• Sample preparation: Adjust sample to 0.1% TFA
• Equilibration & Washing solutions: (0.1% TFA in MilliQ water): 100 µL 1% TFA in MilliQ water, 900 µL MilliQ water
• Elution solution (50% acetonitrile in 0.1% TFA): 300 µL HPLC-grade acetonitrile, 240 μL MilliQ water, 60 µL 1% TFA in MilliQ water
  
  Note: For electrospray, elute with 1% formic acid/50% HPLC-grade methanol

Procedure

NOTE: Resin bed provides back pressure, so set pipettor to 10 µL, depress plunger to dead stop and slowly release or dispense plunger throughout operation.

1. Equilibrate:
   
   Aspirate 10 µL wetting solution into tip and dispense to waste. Repeat. Apsirate equilibration solution into tip and dispense to waste. Repeat.
2. Bind & wash:
   
   Bind peptides to ZipTip pipette tip by aspirating and dispensing 3-7 cycles (simple mixtures), up to 10 cycles (complex). Aspirate washing solution and dispense to waste. Repeat wash once.
   
   Note: A 5% methanol in 0.1% TFA/water wash can improve desalting efficiency.
3. Elute:
   
   Dispense 1-4 µL of elution solution into clean 0.5 mL Eppendorf microcentrifuge tube using a standard pipette tip (Note: if µ-C-18, dispense 0.5-2 µL of elution solution). Aspirate and dispense eluant through ZipTip at least 3 times without introducing air. Sample recovery can be improved by increasing elution volume to 10 µL (but at expense of concentration).