Apr 15, 2020

Public workspaceSample preparation to mass spectrometer using ZipTipC18

DOI
dx.doi.org/10.17504/protocols.io.be45jgy6
  • 1University of Manitoba
Neilier Junior
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DOI: dx.doi.org/10.17504/protocols.io.be45jgy6
Protocol Citation: Neilier Junior 2020. Sample preparation to mass spectrometer using ZipTipC18. protocols.io https://dx.doi.org/10.17504/protocols.io.be45jgy6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 15, 2020
Last Modified: April 15, 2020
Protocol Integer ID: 35709
Abstract
ZipTip is a 10 µL pipette tip that aims to concentrate and purify from femtomols to picomols protein and oligopeptide .

Materials
MATERIALS
ReagentZipTipC18Merck Millipore (EMD Millipore)
ReagentMilliQ Water
ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998
ReagentPierce™ Trifluoroacetic Acid (TFA), Sequencing gradeThermo FisherCatalog #28904
  • Kimwipes

  • P-10 pipettor

  • 1.5 mL microtubes

  • 0,2 mL microtubes
Safety warnings
The contamination of the samples during the preparation is somewhat recurrent and this can compromise the analysis by LC-MS / MS. To avoid contaminants such as saliva and keratin, wear personal protective equipment: mask, gloves and lab coat
Before start
Make sure all reagents and equipment are available. Plan the experiment!
Prepae the materials and workspace
Prepae the materials and workspace
Calibrate the P-10 pipette to 10 μL.
Name 4 microtubes of 1.5 mL with the respective reagents:

  • ACN100% (I)
  • TFA0.1% (I)
  • TFA 0.1% (II)
  • ACN100% (II)

Add 1 mL of the respective solutions to the tubes above.
Name new 0.2 mL microtubes with sample names followed by “zt”.
Aliquot 50 μL of ACN 100% in each “zt” tube.
Fit the ZipTip with the pipette already attached, so as not to pass air through the "chromatographic column". Hold down until you start the procedure with the first pipetting.
Procedure
Procedure
  • Cleaning and preparation: ACN100% (I)

Pipette the acetonitrile from the ACN100% (I) tube 10 times, discarding the contents on the kimwipes


  • Equilibrate: TFA0.1% (I)

Pipette the acetonitrile from the TFA0.1% (I) tube 10 times, discarding the contents on the kimwipes
  • Binding of pepitides (analyte): sample tube

Homogenize the sample with ZipTip 20 times
  • Wash: TFA0.1% (II)

Pipette the acetonitrile from the TFA0.1% (II) tube 10 times, discarding the contents on the kimwipes
  • Elution: ACN100% (II)

Homogenize 20 times in the “zt” tube, already with 50 μL of ACN100%
Shutdown: ACN100% (II)

Pipette the acetonitrile from the ACN100% (II) 10 times, discarding in the “zt” tube.
NOTE
NOTE
During the entire process, no air should enter through ZipTip. Pipetting should be done slowly to maximize chemical interactions