Sample Preparation of Oligonucleotides Prior to MALDI-TOF - Millipore

Technical Note
Sample Preparation of Oligonucleotides Prior
to MALDI-TOF MS Using ZipTip C18 and ZipTip µ-C18
Pipette Tips
iNTroducTioN
ZipTip ® is a 10 µL (P-10) pipette tip with a bed of
chromatography media fixed at its end such that there is
no dead volume. It is intended 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 0.5-4 µL for direct
transfer to a MALDI-TOF MS target or vial.
This protocol provides a guideline for using ZipTipC18 to
desalt oligonucleotide samples prior to MALDI-TOF MS
analysis. C18 is offered in two bed volumes; ZipTipC18 - a
standard bed of 0.6 µL for sample elution in 1 to 4 µL and
ZipTip µ-C18 - a micro bed of 0.2 µL for elution in < 1 µL.
maTerials
• ZipTipC18 or ZipTip µ-C18 pipette tips
• P-10 pipettor, multi-channel P-10 pipettor (Biohit Proline ®
pipettor recommended) or compatible automated liquid
handling work station
• Wetting solution: 50% acetonitrile (ACN)/Milli-Q ® water
• Equilibration solution: 0.1 M triethylammonium acetate
(TEAA), pH 7.0
• Wash solution #1: 0.1 M triethylammonium acetate
(TEAA), pH 7.0
• Wash solution #2: Milli-Q water
• Elution buffer: 50% ACN/Milli-Q water
For direct spotting in matrix, elute with 3-hydroxypicolinic
acid in 50% acetonitrile containing ammonium citrate
matrix composition:
• 9 parts 50 mg/mL 3-hydroxypicolinic acid in 50%
acetonitrile/Milli-Q water.
• 1 part 50 mg/mL ammonium citrate in Milli-Q water
NOTE: Since the resin bed provides a slight back-pressure, the pipette should
not be used as an accurate volumetric dispenser. To achieve optimal sample
uptake and delivery, set pipettor to 10 µL and attach tip securely. Depress
plunger to dead stop and slowly release or dispense plunger throughout
operation.

Procedure
Prepare the sample:
Optimal binding and recovery of oligonucleotides from C18 is
performed in the presence of TEAA, an ion-pairing agent.
Dissolve or resuspend oligonucleotide in 10 µL of the
equilibration solution. For oligonucleotide synthesis
products, add 100-200 ng of oligonucleotide to 10 µl of the
equilibration solution.
NOTE: For synthetic oligonucleotides, short termination sequences will be
lost if acetonitrile concentrations in the binding mixture exceed 10%. If this
is the case, dilute with 0.1 M TEAA as described above.
equilibrate the ZipTip for sample Binding:
1. Prewet the tip by depressing pipettor plunger to a dead
stop using the maximum volume setting of 10 µL.
Aspirate wetting solution into tip. Dispense to waste.
Repeat.
2. Equilibrate the tip for binding by washing with 10 µL of
equilibration solution 3 times.
Bind and Wash the oligonucleotides:
Follow these steps after equilibration:
1. Bind oligonucleotides to ZipTipC18, by fully depressing the
pipettor plunger to a dead stop. Aspirate and dispense
sample 5 to 10 cycles.
2. Wash tip 3 times with 10 µL of fresh wash solution #1,
dispensing to waste after each aspirate and dispense
cycle.
3. Wash tip 3 times with 10 µL of fresh wash solution #2,
dispensing to waste after each aspirate and dispense
cycle.
elute the oligonucleotides:
For ZipTipC18, dispense 1 to 4 µL of elution solution into a
clean vial using a standard pipette tip. In the case of
ZipTip µ-C18, dispense 0.5 to 2 µL of elution solution into a
clean vial. Carefully, aspirate and dispense eluant through
ZipTip at least 3 times without introducing air. Sample
recovery can be improved (at the expense of concentration)
by increasing elution volume up to 10 µL.
CAUTION: Acetonitrile is volatile and evaporation can occur rapidly.
If this occurs, add more eluant to recover sample.
www.millipore.com
direct spotting onto maldi-ToF ms Target:
Elute with matrix in elution solution.
1. Follow the above elution procedure.
2. Aspirate desired volume of the desalted-concentrated
sample into ZipTip and dispense directly onto the target.
resulTs
Counts x 100
Counts x 100
18
16
14
12
10
8
50
40
30
20
10
4 6 8 10 12 14 16
Mass (m/z x 1,000)
3393.34
4316.5
6779.43
8602.63
Direct
Spotting
After
ZipTip
12981
4 6 8 10 12 14
Mass (m/z x 1,000)
MALDI-TOF MS spectra of 22, 28 and 42mer oligonucleotides. Sample
consisted of 50 ng of each oligonucleotide in 10 µL water containing
(Na) 4 EDTA (10 mM), MgCl 2 (1.0 mM), NaCl (100 mM) and glycerol (10% w/v).
Spectra were taken before and after desalting with ZipTip C18.
To Place aN order or
receiVe TecHNical assisTaNce
In the U.S. and Canada, call toll-free 1-800-MILLIPORE (1-800-645-5476)
Outside of North America, please visit www.millipore.com/offices.
For Technical Service, please visit www.millipore.com/techservice.
Millipore, ZipTip, Milli-Q and Advancing Life Science Together are
registered trademarks of Millipore Corporation.
The M mark is a trademark of Millipore Corporation.
Biohit Proline is a registered trademark of Biohit Oyj.
Lit. No. TN225 Rev. B 10/10 Printed in U.S.A. LS-SBU-10-03805
© 2010 Millipore Corporation, Billerica, MA 01821 U.S.A.
All rights reserved.