Technical Note Sample Preparation of Oligonucleotides Prior to MALDI-TOF MS Using ZipTipC18 and ZipTipµ-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 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 ZipTipµ-C18 - a micro bed of 0.2 µL for elution in < 1 µL. better data quality. The sample is aspirated and dispensed materials through ZipTip to bind, wash, and elute. Recovered samples • ZipTipC18 or ZipTipµ-C18 pipette tips are contaminant-free and eluted in 0.5-4 µL for direct • P-10 pipettor, multi-channel P-10 pipettor (Biohit Proline® transfer to a MALDI-TOF MS target or vial. 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 Direct Spotting onto MALDI-TOF MS Target: Prepare the Sample: Elute with matrix in elution solution. Optimal binding and recovery of oligonucleotides from C18 is 1.Follow the above elution procedure. performed in the presence of TEAA, an ion-pairing agent. 2.Aspirate desired volume of the desalted-concentrated Dissolve or resuspend oligonucleotide in 10 µL of the sample into ZipTip and dispense directly onto the target. equilibration solution. For oligonucleotide synthesis products, add 100-200 ng of oligonucleotide to 10 µl of the Results 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. 1.Prewet the tip by depressing pipettor plunger to a dead Counts x 100 Equilibrate the ZipTip for Sample Binding: Direct Spotting 18 stop using the maximum volume setting of 10 µL. 16 14 12 10 Aspirate wetting solution into tip. Dispense to waste. 8 Repeat. 4 2.Equilibrate the tip for binding by washing with 10 µL of 6 8 10 12 14 16 Mass (m/z x 1,000) equilibration solution 3 times. Bind and Wash the Oligonucleotides: 6779.43 Follow these steps after equilibration: 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, Counts x 100 1.Bind oligonucleotides to ZipTipC18, by fully depressing the After ZipTip 50 3393.34 40 30 8602.63 4316.5 20 12981 10 4 6 8 10 12 14 Mass (m/z x 1,000) dispensing to waste after each aspirate and dispense cycle. Elute the Oligonucleotides: For ZipTipC18, dispense 1 to 4 µL of elution solution into a 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), MgCl2 (1.0 mM), NaCl (100 mM) and glycerol (10% w/v). Spectra were taken before and after desalting with ZipTipC18. clean vial using a standard pipette tip. In the case of ZipTipµ-C18, dispense 0.5 to 2 µL of elution solution into a clean vial. Carefully, aspirate and dispense eluant through recovery can be improved (at the expense of concentration) TO PLACE AN ORDER OR RECEIVE TECHNICAL ASSISTANCE by increasing elution volume up to 10 µL. In the U.S. and Canada, call toll-free 1-800-MILLIPORE (1-800-645-5476) ZipTip at least 3 times without introducing air. Sample CAUTION: Acetonitrile is volatile and evaporation can occur rapidly. If this occurs, add more eluant to recover sample. www.millipore.com 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.