Technical Note - Millipore

Date:
Lit. No.:
Title:
February 2, 2001
TN233
Technical Note
A Publication of Technical Services
Peptide Sample Preparation Prior to MALDI-TOF and Electrospray MS
Using ZipTipSCX Pipette Tips
INTRODUCTION
ZipTip® is a 10 µL pipette tip with a bed of chromatography media fixed at its end. It is designed
for purifying and concentrating femtomoles to picomoles of protein, peptide or oligonucleotide
samples prior to analysis providing better data quality. The sample is aspirated and dispensed
through ZipTip to bind, wash, and elute. Recovered samples are contaminant-free and eluted in
1-4 µL for direct transfer to a MALDI-TOF MS target or vial.
These instructions describe the use of ZipTipSCX pipette tips containing strong cation exchange
resin for the concentration of peptides and removal of salts and nonionic detergents from
peptide mixtures prior to mass spectrometry.
Strong cation exchange (SCX) chromatography is an alternative to reversed-phase (C18)
chromatography for the separation of peptides. SCX chromatography works especially well for
tryptic peptides that have a minimum of two positive charges per molecule at acidic pH.
Peptides from acidic buffer are captured on the ZipTip resin, washed, and then eluted directly
onto a MALDI-TOF MS target at basic pH. Once the samples have dried, they are overlaid with
matrix, dried again, and analyzed.
ZipTipSCX pipette tips effectively capture dilute peptides from in-gel or in-solution digested protein
samples and reversed-phase chromatography fractions containing up to 45% acetonitrile.
ZipTipSCX pipette tips can also be used for the removal of non-ionic or zwitterionic detergents.
MATERIALS
NOTE: For optimal results all solutions should be freshly prepared using Milli-Q ® grade water.
• ZipTipSCX pipette tips
• 10 µL pipettor - VistaLab ULTRON Handheld Liquid Handling System with ZipTip Adapter Kit
(cat. no. 1052-9016) is recommended as a variable speed, programmable electronic pipettor.
The Biohit Proline® electronic multi-channel pipettor is recommended for processing eight
samples simultaneously. Visit millipore.com/ziptip for a current listing of compatible automated
liquid handling work stations
• Sample Preparation Solution: 10% trifluoroacetic acid (TFA) in Milli-Q grade water
• Washing Solution: 0.1% TFA in Milli-Q grade water. Washing Solution for detergent removal: 0.1%
TFA / 30% methanol in Milli-Q grade water
• Elution Solution: 5% ammonium hydroxide/30% methanol in Milli-Q grade water
In North America call toll free 1-800-Millipore (645-5476)
In Europe fax +33 3.88.38.91.95
In Japan call (03) 5442-9716
In Asia call (852) 2803-9111
In Australia call 1 800 222 111
In all other countries call (781) 533-8622 www.millipore.com/ziptip

PROCEDURE
NOTE: Because the adsorptive bed provides a slight back pressure, do not use the ZipTip pipette tip for
accurate volumetric dispensing. To achieve optimal sample uptake and delivery, set the pipettor to 10 µL
and attach the ZipTip pipette tip securely. Throughout the procedure, depress and release the plunger
slowly to ensure optimal movement of solution through the resin bed.
Preparing the Sample:
Dissolve the sample in 5-100 µL of Washing Solution. For in-solution mixtures, add the appropriate
amount of Sample Preparation Solution to achieve a final TFA concentration of 0.1%.
Equilibrating the ZipTipSCX Pipette Tip
1. Set the pipettor volume to 10 µL.
2. Depress the plunger to dead stop and aspirate the Washing Solution into the ZipTip pipette tip,
dispense to waste.
3. Repeat three times.
Binding and Washing
1. Bind peptides to the equilibrated ZipTip pipette tip, by performing 10-15 aspirate-dispense
cycles of the entire sample.
2. Aspirate 10 µL of Washing Solution into the ZipTip pipette tip, dispense to waste. Repeat five
times to ensure sufficient removal of salts and detergents.
Eluting the Sample for MALDI-TOF MS
1. Desorb bound peptides by aspirating 2.5 µL of Elution Solution into the ZipTipSCX pipette tip.
Spot directly onto MALDI-TOF MS target, pipette up and down three times.
2. Dry spots.
3. Overlay each peptide spot with 1 µL of preferred matrix solution.
Eluting the Sample for Electrospray MS
1. Desorb bound peptides by aspirating 5 µL of Elution Solution into the ZipTip pipette tip. Elute
into clean tube. Pipette up and down three to five times.
2. Allow sample to evaporate to near dryness.
3. Reconstitute sample in compatible electrospray solution.
Results
Figure 1. In-solution Protein Digest Purification. 80 fmol of BSA digest in 0.1% TFA / 20%
acetonitrile was concentrated and purified with ZipTipSCX. The MALDI-TOF MS spectrum was
obtained on an ABI Voyager -DE Biospectrometry Workstation. Database search of the
resulting peptide map successfully identified the protein with 28% coverage.

Direct Spot
Results
Figure 2. Detergent removal from BSA digest. A 100 fmol/µL BSA was digested with trypsin. For
direct spot, 1µL of digest was mixed with 1µL of 1% detergent solution and spotted onto a
MALDI plate. Another 1µL of BSA digest (100 fmol) was mixed with 1 µL of 10% detergent
solution and 18 µL of 0.1% TFA. The peptides were captured on ZipTipSCX, washed with 0.1%
TFA/30% methanol and eluted with 2.5 µL of 5% ammonia/30% methanol directly onto a
MALDI plate. All spots were dried, overlaid with matrix and analyzed on an ABI Voyager-DE
Biospectrometry Workstation. (A) 0.5% Tween-20, (B) 0.5% Triton X100, (C) 0.5% CHAPS.
Millipore, ZipTip and Milli-Q are trademarks of Millipore Corporation or an affiliated company.
Biohit Proline is a registered trademark of Biohit Oy.
ULTRON is a trademark of VistaLab Technologies, Inc.
Tween-80 is a registered trademark of the Atlas Powder Company.
Triton is a registered trademark of the Rohm and Hass Company
Voyager DE and Biospectrometry are trademarks of ABI.
Copyright © 2002 Millipore Corporation, Bedford, MA
A
B
C
After ZipTipSCX