Millipore's ZipTip Cures Small Sample Prep Woes

Preparing tiny samples of biomolecules for instrumental analysis taxes the creative capabilities of most biologists. Samples going into mass spectrometers, in particular, require extra care. I've seen all sorts of gizmos used for this kind of sample prep—capillaries filled with desalting media, Pasteur pipettes plugged with alternating layers of cotton and ion exchange resins, etc. Most makeshift methods require adding buffer to recover the sample, thereby diluting the sample and sometimes adding another (concentration) step. Now there is a better way.

The new ZipTip sample preparation system from Millipore Corp. can be used to concentrate, desalt, and remove detergents from biological samples. ZipTip consists of a 10 µL pipette tip with approximately a half microliter bed of resin fixed at its end. ZipTipC₁₈, the first of what we hope will be many products in the ZipTip series, desalts femtomoles of peptides in less than 60 seconds. Elution in 1 - 4 µl of acetonitrile/water simplifies direct transfer to a mass spectrometer.

ZipTipC₁₈

In operation, ZipTip is placed on a standard single or multi-channel pipettor. Sample is aspirated and dispensed through the resin bed to bind, wash and elute peptides and small proteins. Supported by protocols developed at Millipore, ZipTipC₁₈ saves time by providing a fast, ready to use device for offline sample preparation.

Features and Benefits of ZipTipC₁₈

• Pipette tip device format provides a simple, easy to use Sample preparation and precise liquid handling in one device
• C₁₈, 15 µm beads assure rapid sample processing. According to Millipore samples are purified in sixty seconds
• Robust: well-characterized interaction Elution in solvent/matrix for direct delivery to a MALDI target
• Suitable for use with single, multi-channel and automated liquid handling systems
• Flexible Increased throughput saves time
• Small 0.6 microliter chromatographic bed volume is suitable for minute sample volumes and masses Minimal sample loss

How it Works

The following protocol is used to prepare samples using ZipTipC₁₈ for sample binding, washing, and elution of peptides or small proteins (< 40 Kd) for MALDI MS, Electrospray MS, or other analytical techniques.

Materials: ZipTipC18 contains C18 spherical silica (15 µm , 200Å pore size) in a 0.6 µL bed volume; ZipTipC₁₈ pipette (single or multi-channel); wetting buffer: 50% acetonitrile in water; equilibration buffer: 0.1% triflouroacetic acid (TFA) in water; elution buffer: 50% acetonitrile in water. For direct MALDI, prepare matrix in 50% acetonitrile/water.

NOTE: Since the adsorptive bed provides back pressure, do not use ZipTip for accurate volumetric dispensing. To achieve optimal performance, adjust the P-10 pipette to 10 µL and press ZipTip on securely. Depress plunger to dead stop to allow maximum suction and sample dispensing.

Multiple pipette ZipTips save time by processing multiple samples

Protocol: Use the ZipTip with samples containing picogram to micrograms of sample. The absorption efficiency of ZipTip is sample- and concentration- dependent. Maximum binding to the ZipTip is achieved in the presence of TFA or other ion-pairing agents. To minimize sample dilution and enhance analyte binding, use 10% TFA to achieve a final concentration of 0.1% TFA. In the case of excess detergent, dilute sample with 0.1% TFA to achieve acceptable binding conditions, for example, SDS (< 0.1%), Triton (< 1%), and Tween (< 0.5%).

To equilibrate the ZipTipC₁₈ for Sample Binding

1. Prewet the tip by depressing plunger to a dead stop using the maximum volume setting of 10 µL. Aspirate Wetting buffer into tip. Dispense to waste. Repeat.
2. Equilibrate the tip for binding by washing it twice with the Equilibration buffer. (See "Materials" section for details.)

To bind, wash, and elute peptides after ZipTip equilibration:

1. Bind peptides and proteins to ZipTipC₁₈ by fully depressing the pipette plunger to a dead stop. Aspirate and dispense sample 5 to 10 cycles depending on sample concentration. Dilute solutions require increased contact time.
2. Wash tip and dispense to waste using two cycles of 0.1% TFA, water, or 5% methanol in 0.1% TFA/water mixture. You may require additional washing for electrospray applications or samples containing high salt or detergent.
3. Elute by dispensing 2 to 4 µL of Elution buffer to clean vial using a standard pipette tip. Carefully, aspirate and dispense eluant through ZipTip at least three times without pushing air into the sample. For Electrospray MS, elute sample into clean vial and apply directly for analysis. For MALDI MS, elute with or without matrix in 50% acetonitrile/water.

   CAUTION: Acetonitrile is volatile and evaporation can occur rapidly. If this occurs, add more eluant to recover sample.

   Pipette 1 to 2 µL of desalted-concentrated sample directly onto target by depressing plunger until appropriate volume is dispensed. Save or discard the remaining sample with tip.

   NOTE: For samples containing 1 µg of protein or 0.5 µg of peptide, you can achieve maximum recovery by increasing elution volume to 10 µL or by performing multiple elutions (2 × 5 µL). For information on fractionating a peptide, see the "Technical Assistance" section.

   Protein recovery yields using ZipTip are excellent

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   Table 1. Reagent compatibility of ZipTipC₁₈

   Compatibility code:
   YES - Long exposures at room temperature have no significant effect.
   ? - Questionable - Short exposures at room temperature cause little or no damage.
   No - Not recommended - Short exposure may cause permanent damage.

   Acetic Acid (Glacial)	YES
   Acetone	NO
   Mercaptoethanol (0.1M)	YES
   Acetonitrile (100%)	YES
   Aliphatic Esters	?
   Ammonium Hydroxide (5%)	YES
   Benzene (100%)	NO
   Butyl Acetate	NO
   Chloroform (1%)	?
   Dichlorobenzene (100%)	NO
   Dichloromethane (1%)	?
   Diethanolamine (5%)	YES
   Dimethyl Acetamide (100%)	NO
   Dimethyl Formamide (1%)	YES
   Dimethyl Formamide (100%)	NO
   Ethanol (100%)	YES
   Formic Acid (5%)	YES
   Guanidine HC1 (6 M)	YES
   Hydrochloric Acid	YES
   Hydrogen Peroxide	YES
   Isopropyl Alcohol (100%)	YES
   Mercaptoethanol (1.0M)	?
   Methyl Alcohol (100%)	YES
   Methyl Ethyl Ketone (100%)	NO
   Methyl Isobutyl Ketone (100%)	YES
   n-Butanol (100%)	YES
   Nitric Acid (0.1N)	YES
   Nitric Acid (1.5N)	YES
   Phenol (0.5%)	NO
   Phosphoric Acid (1M)	YES
   Sodium Azide (1%)	YES
   Sodium Hydroxide (0.1N)	YES
   Sodium Hydroxide (0.5N)	YES
   Sodium Hypochlorite (100 ppm)	YES
   Sodium Hypochlorite (200 ppm)	YES
   Sulfuric Acid (1%)	YES
   Toluene (1%)	NO
   Triton X-100	YES
   Tween	YES
   Urea (6M)	YES

   Triton X-100 is a registered trademark of Union Carbide Chemical and Plastics, Inc. Tween is a registered trademark of ICI Americas, Inc.

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   Specifications

   Materials of Construction: Pipette tip: Polypropylene Resin: C18 spherical silica (15 µm, 200Å pore size)
   Tip capacity: 10 µL Adsorptive bed: 0.6 µL Length: 31 mm Max. capacity: 2 µg when used with saturating amounts of analyte
   Max. Temperature: 70°C Min. Temperature: 4°C
   Working pH Range: 2 to 13.

   By Angelo DePalma

   For more information: Donna Murphy, Technical Services, Millipore Corp., 80 Ashby Road, Bedford, MA 01730-2271. Tel: 781-533-8244.

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