Purification and Concentration of Peptides Isolated

From In-gel Digestion of Protein Bands by Zip-Tip

(Taken from: Advanced Protein Technology Centre, Hospital for Sick Children, Toronto, Canada)
As it would be in the case of Reversed-Phase HPLC, an important precaution to take during the procedure
described below is never to allow the Zip-Tip column to dry out during any one of the steps. Channeling due to
air-bubbles in the Zip-Tip will lead to poor retention of peptides, causing large losses of peptides, and also a
great variability in yields of individual peptides.
(The procedure described here may be used in parallel to process several digests side-by-side.)
Items Required For Each Peptide Digest:
Zip-Tip C18 Cartridge column
Tube A.

100l Acetonitrile (HPLC Grade) - Wetting Solution

Tube B.

1000l water (HPLC Grade) - Cleaning Solution

Tube C.

10-20l Sample Solution

Dissolve the extracted peptides in 10-20l 0.5% formic acid; make sure to wash the lower sides of
the PCR tube containing the extracted peptides to achieve maximum dissolution of the peptides.

Tube D.

500l 0.5% Formic acid - Desalting/Wash Solution

Tube E.

100l 0.5% Formic Acid in 1:1 (v/v) water : acetonitrile - 1st Extraction Solution.

Tube F.

100l Acetonitrile - 2nd Extraction Solution

Tube G.

A clean empty 200l PCR tube

1.

Attach the Zip-Tip column to the 20l micropipet (e.g. Gilson's Pipetman P20) snugly. Adjust the volume
setting to 7l. Carefully, withdraw acetonitrile (Tube A) through the Zip-Tip and then, while the tip of the
Zip-Tip is still under acetonitrile, pipet out the acetonitrile carefully, taking precaution to prevent introducing
air bubbles into the Zip-Tip. Repeat this step at least 7-8 times, or until no bubbles arise during the pipetting
out step. Finally, pipet out the acetonitrile slowly, and while the plunger is still down, immerse the tip of the
Zip-Tip into the water (Tube B).

2.

Slowly withdraw 7l of water through the Zip-Tip, and then pipet it out carefully, taking care not to introduce
any air into the Zip-Tip. Repeat this step carefully at least 10 times to ensure that all acetonitrile has been
washed away.

3.

Pipet out the water, and while the plunger is still down, move the tip of the Zip-Tip into the sample solution
(Tube C). Carefully, fill the Zip-Tip with the Sample Solution, and, slowly push out into the tube (Tube C).
Repeat at least 10 times to ensure that most of the peptides have been retained on the Zip-Tip.

4.

In the same fashion as in steps 2, and 3, above, wash the Zip-Tip with 0.5% formic acid solution (Tube D) at
least 10 times to perform the desalting and washing of the peptides.

5.

After pipeting out the wash solution and, with the plunger is still down, transfer the Zip-Tip to the tube
containing the extraction solution (Tube E), and slowly fill with the extraction solution. Wait for 20 sec to
ensure a complete extraction.

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6.

Pipet out the extracting solution (Extract 1) into the empty tube G, and, while the plunger is still down, move
to the tube F containing the acetonitrile. Slowly withdraw acetonitrile through the Zip-Tip.

7.

After waiting for 10 sec., pipet out the solution (Extract 2) into the tube G containing the Extract 1.

8.

Submit the combined extracts (Tube G) for MS Analysis, or store in a freezer until required for analysis. If
you intend to us by mail, then we recommend that you dry the solution completely in a Speed-vac before
mailing.

A Checklist For the Zip-Tip Protocol

Reagent

Digest 1
___________

Digest 2
___________

Digest 3
___________

Digest 4
___________

Digest 5
___________

Wetting Step
Repeat 7-8X, or
until
no
air
bubbles in the
column
Washing Step
Perform
washings 10X
Sample Loading
Apply at least
10X
Desalting/Wash
Perform at least
10 washings
Extraction 1
---------------------Extraction 2

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Sample Clean up Using Zip-Tips in Preparation for MALDI-TOF Mass Analysis:

Zip-Tips are pipette tips that contain immobilized C18, C4 or some other resin attached at their very tip
occupying about 0.5l volume. For most purposes we use C18 resin. Our usual protocol is:
1. Use a P20 pipetter set to 10l for Zip-Tips
2. Wash the Zip-Tip with 0.1% trifluroacetic acid (TFA) in acetonitrile
3. Wash the Zip-Tip with 0.1% TFA in 1:1 acetonitrile:water
4. quilibrate the Zip-Tip twice with 0.1% TFA in water
5. The sample, typically dissolved in 10 l of 0.1% TFA, is passed through the Zip-Tips repeatedly
by pipeting in and out to bind the sample to the resin.
6. Wash the Zip-Tip three times with 0.1% TFA, 5% methanol in water
7. Elute the sample from the Zip-Tip in 1.8l of matrix, typically alpha-cyano-4-hydroxycinnamic
acid in 0.1% TFA 50% acetonitrile, directly on the MALDI-TOF sample plate.

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Zip Tip Protocol for Peptide and Protein Analysis

I. Materials
Wetting solution (100% HPLC-grade acetonitrile)
Stock solution (1% TFA in MilliQ water): 990 L MilliQ water, 10 L TFA
Sample preparation: Adjust sample to 0.1% TFA
Equilibration & Washing solutions: (0.1% TFA in MilliQ water):
100 L 1% TFA in MilliQ water, 900 L MilliQ water
Elution solution (50% acetonitrile in 0.1% TFA):
300 L HPLC-grade acetonitrile, 240 L MilliQ water, 60 L 1% TFA in MilliQ water
Note: For electrospray, elute with 1% formic acid/50% HPLC-grade methanol
II. Procedure
NOTE: Resin bed provides back pressure, so set pipettor to 10 L, depress plunger to dead stop and slowly
release or dispense plunger throughout operation.
1. Equilibrate:
Aspirate 10 L 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 L of elution solution into clean 0.5 mL Eppendorf microcentrifuge tube using a standard
pipette tip (Note: if -C-18, dispense 0.5-2 L 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 L (but at expense of concentration).

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ZipTip cleanup of samples

We use Millipore ZipTips, micro-C18, part no. ZTC18M096; and Hamilton syringes, 1701N (10 L) and
1710N (100 L) (or 1701RNR and 1710RNR) (blunt needles, as used in HPLC). Note: The manufacturer
of the ZipTips suggests using a pipetter, which works, but peptide yield is 2-3x better if you use a syringe.
You will need the following solutions:
1) 0.1 % TFA in good quality water
2) 50/50 mixture of solution 1 and acetonitrile (50/50).

Procedure:
Prepare the sample: Extracts from the gel digest will contain about 50-100 L of ~50% acetonitrile
(ACN). Reduce the volume to about 5 L in a vacuum centrifuge (Speedvac). This concentrates the sample
but also reduces the ACN concentration to <5%. It is better not to allow the samples to go to dryness
because sample loss will occur, but if a sample goes dry, reconstitute with 5 L of 50/50. (Samples can be
stored at this point at +4, -20 or -80C. (ACN and water separate into two phases at -20C, but for these
small samples, it doesnt seem to matter.)
Condition the tip: Rinse the tip with 50/50, and then equilibrate with 0.1% TFA. We prefer to do a single
100 L rinse with each of the two solutions, using a 100 L syringe. At the very start of the 50/50 wash,
tap the syringe plunger and squirt through a bit of solvent to get rid of air bubbles.
Adsorb: Dilute the sample with 90 L of 0.1% TFA. This is to reduce the concentration of ACN so that the
peptides will bind to the C18 resin in the ZipTips. Attach the conditioned ZipTip to a 100 L syringe (just
press on) and draw the sample solution back and forth through the ZipTip at least ten times. Do this rather
slowly to allow adsorption to take place. Keep the tip wet: dont allow air bubbles to get trapped in the tip.
Rinse: Empty a syringe with ~100 L of 0.1% TFA slowly through the tip to rinse out salts and other
water-soluble impurities.
Elute: There are two ways to do this.
1. Put 3 L of 50/50 into a 200 L PCR tube. With a 10 L pipetter or syringe, draw the solution slowly
back and forth through the C-18 resin at least 5-10 times to improve recovery.
2. Put 4 L of 50/50 in a 10 L syringe. Put the tip on the syringe and elute slowly. Discard the first one
L, then collect the rest. Do the elution slowly to allow desorption to take place.
Store eluted samples at -20 or -80C. They should be analyzed as soon as possible after elution, because
peptides tend to irreversibly adsorb to the tube walls or otherwise vanish.

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Millipore ZipTip
(For Reversed-Phase)
Materials
ZipTipC18 tips- a standard bed of 0.6 L for sample elution in 1 to 4 L. Appropriate for peptides and
low molecular weight proteins.
ZipTipC4 tips- a micro bed of 0.2 L for sample elution in <1 L. Appropriate for low to intermediate
molecular weight proteins, proteins over 100,000 Da.
The procedure also requires a compatible 10 L pipettor. For simultaneous processing of multiple
samples, Millipore recommends the Biohit ProlineTM Multi-channel Pipettor.

Solutions (make fresh daily):

Wetting solution

ZipTipC18 tips

ZipTipC4 tips

100% Acetonitrile (ACN)

Sample Prep

100% Acetonitrile (ACN)

0.5% TFA in ddH2O

0.5% TFA in ddH2O

(optional) Guanidine-HCl 1-6 M maybe added

Equilibration solution

0.1% TFA in ddH2O

0.1% TFA in ddH2O

Wash solution

0.1% TFA in ddH2O

0.1% TFA in ddH2O

Elution solution

0.1% TFA/ 50% ACN

0.1% TFA/ 50%ACN

With or without Matrix With or without Matrix

Procedure
ZipTipC18 tips
Maximum binding occurs in the presence of TFA (0.1%) or other ion-pairing agents. To maximize analyte
binding, use the appropriate sample preparation solution. Ensure that the final solution has a pH < 4.

ZipTipC4 tips
Optimal binding may require a chaotropic agent (guanindine-HCl at a final concentration of 1-6 M). If the
sample does not already contain chaotropic salts, add them a few minutes prior to binding. These salts will
be removed during the wash step following binding.
Equilibrate the ZipTip pipette tip for sample binding
Depress pipettor plunger to a dead stop. Using the maximum volume setting of 10 L, aspirate wetting
solution into the tip. Dispense to waste. Repeat 2x.
1.

Aspirate equilibration solution. Dispense to waste. Repeat 2x.

Bind and wash the peptides or proteins

Follow these steps after equilibrating the ZipTip pipette tip:

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1.

Bind peptides and/ or proteins to ZipTip pipette tip by fully depressing the pipette plunger to a dead stop. Aspirate
and dispense the sample 7-10 cycles for maximum binding of complex mixtures.

2.

Aspirate wash solution into tip and dispense to waste. Repeat 4x.
NOTE: 5% methanol in 0.1% TFA/ water wash can improve desalting efficiency. Additional washing may be
required for electrospray MS.

Elute the peptides or proteins

For ZipTipC18 (standard bed format) and ZipTipC4, pipette tips, dispense 1-4 L of elution solution into a clean vial using a
standard pipette tip. In the case of ZipTip -C18 (micro bed format) pipette tips, dispense 0.5-2 L of elution solution into a
clean vial.
Caution: Acetonitrile and methanol are volatile and evaporation can occur
rapidly. If this occurs, add more eluant to recover sample.
Carefully, aspirate and dispense eluant through ZipTip pipette tip at least three times without introducing air into the sample.
Sample recovery can be improved (at the expense of concentration) by increasing the volume to 5 L.
For direct spotting onto a MALDI-TOF MS target
Elute with or without matrix elution solution.
1.

Pipette 0.5-4 L of desalted-concentrated sample directly onto target by depressing plunger until appropriate volume
is dispensed.

2.

Save or discard the remaining solution.

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Introduction
ZipTip is a 10 L (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 L for direct transfer to a mass spectrometer target or vial.
This protocol provides a guideline for using ZipTipC18 and ZipTipC4 to facilitate protein or peptide
binding, salt and detergent removal, and sample elution for direct electrospray/nanoelectrospray MS
analysis. C18 is offered in two bed volumes: ZipTip C18 (a standard bed of 0.6 L for sample elution in 1 to 4 L) and ZipTip -C18
(a micro bed of 0.2 L for elution in < 1 L).

Material

ZipTip C18, ZipTip -C18 or ZipTip C4 pipette tips

P-10 pipettor, multi-channel P-10 pipettor (Biohit Proline pipettor recommended) or compatible automated liquid
handling work station

Wetting solution: 50% methanol (MeOH) in (TFA) in Milli-Q water (i.e., water from a Milli-Q water purification
system from Millipore)

Equilibration and washing solution: 0.1%TFA in Milli-Q water

Sample preparation solution: 2.5% TFA in Milli-Q water (5X stock solution)
NOTE: in the case of proteins a 8M guanidine and 2.5% TFA in Milli-Q water solution (5X stock solution) may
substituted if sample solubility is a problem

Elution solution:
Peptides - 50% MeOH/0.1% TFA or 0.1% formic acid in Milli-Q water
Proteins - 75% MeOH/0.1% TFA or 0.1% formic acid in Milli-Q water

NOTE: Since the resin bed provides a slight backpressure, the pipettor should not be used as an accurate volumetric dispenser.
To achieve optimal sample uptake and delivery, set pipettor to 10 L and attach tip securely. Depress plunger to dead stop and
slowly release or dispense plunger throughout operation.

Guidelines For Selecting ZipTipC18 and ZipTipC4

ZipTip C18 or ZipTip -C18 are most applicable for low molecular weight proteins and peptides, while ZipTip C4 is most suitable for
low to intermediate molecular weight proteins. In many cases, the two devices can be used interchangeably, as indicated by the
overlapping bars in the above figure. Because higher molecular weight proteins tend to adsorb tenaciously to hydrophobic
surfaces, ZipTip C4 is recommended for proteins over 100,000 MW.

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Procedure
Prepare the Sample:
Maximum binding to the ZipTip is achieved in the presence of TFA or other ion-pairing agents. To maximize analyte binding,
use the appropriate sample preparation solution. The final TFA concentration should be between 0.1%1.0% at a pH of <4.
Optimal binding of protein to ZipTip may also require a chaotropic agent (e.g. guanidine-HCl at a final concentration of
approximately 14M). If sample does not already contain a chaotropic salt, add a few minutes before binding. 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%).
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 L. Aspirate wetting
solution into tip. Dispense to waste. Repeat.
2. Equilibrate the tip for binding by washing with the equilibration solution 3 times.
Bind and Wash the Peptides or Proteins:
Follow these steps after equilibration.
1. Bind peptides and proteins to ZipTip by fully depressing the pipettor plunger to a dead stop. Aspirate and dispense sample 3
to 7 cycles for simple mixtures and up to 10 cycles for maximum binding of complex mixtures.
2. Wash tip and dispense to waste using at least five cycles of wash solution. A 5% methanol in 0.1% TFA/water wash can
improve desalting efficiency.
Elute the Peptides or Proteins:
For ZipTip C18 and ZipTip C4, dispense 1 to 4 L of elution solution into a clean vial using a standard pipette tip. In the case of
ZipTip -C18, dispense 0.5 to 2 L of elution solution into a clean vial. Carefully, aspirate and dispense eluant through ZipTip at
least three times without introducing air. Sample recovery can be improved (at the expense of concentration) by increasing
elution volume to 10 L or by performing multiple elutions.
CAUTION: Methanol is volatile and evaporation can occur rapidly. If this occurs, add more eluant to recover sample.
For Electrospray MS:
Sample can be eluted into clean vial or, using a GELoader tip (Eppendorf cat. no. 0030 001 222), into a nanospray needle.
1. Cut the GELoader tip about 23 mm above where the tip is fused to its capillary end.
2. Before the final dispense, firmly press the cut-down GELoader tip onto the ZipTip with a slight twisting motion. The leakfree fit allows direct elution into a nanoelectrospray needle.

RESULTS

Nanoelectrospray-ES of lysozyme desalted on ZipTip. ZipTip C18 treatment of salt, guanidine and phosphate buffer
containing lysozyme is shown using nanoelectrospray technology. The charge to mass spectra (right) is devoid of salt
and exhibits excellent resolution. The deconvoluted mass peak is of high quality and accurate mass due to efficient
salt removal.

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What is the binding capacity of ZipTipC18, ZipTip-C18 and ZipTipC4?

ZipTip Capacity: (when used with saturating amounts of analyte)

ZipTipC18: greater than or equal to 1 g. Typically 5.0 g
ZipTip-C18 : Typically 3.3 g
ZipTipC4: greater than or equal to 0.5 g. Typically 3.3 g
C18 is offered in two bed volumes:
ZipTipC18 a standard bed of 0.6 L for sample elution in 1 to 4 L
ZipTip-C18 a micro bed of 0.2 L for sample elution in < 1 L.

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