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USER GUIDE
Pierce™ P
eptide Desalting Spin Columns
Catalog Numbers 89851 and 89852
Doc. Part No. 2162704 Pub. No. MAN0017280 Rev. A.0
Contents
Product Cat. No. Contents Storage
25 spin columns containing 20 mg of resin in a
1:1 water/DMSO slurry
50 spin columns containing 20 mg of resin in a
1:1 water/DMSO slurry
Store at 2-8°C.
Pierce™ P
89852
eptide Desalting Spin Columns
89851
Product description
The Thermo Scientic™ Pierce™ P
The polymer-based hydrophobic resin contained in the column is stable from pH 1-14 and binds peptide samples with excellent recovery (typical
recovery is >90%) under reversed phase conditions. Columns can also be used for removal of excess hydrolyzed/quenched TMT reagents after
labeling to enable ecient and reliable downstream LC-MS analysis.
Procedure summary
Proteolytic digests of proteins extracted from cells or tissues are loaded onto an equilibrated desalting spin column. Peptides are bound to the
hydrophobic resin under acidic aqueous conditions and desalted by washing the column with acidied water by low-speed centrifugation. A
solution with acidied organic solvent (0.1% TFA, 50% acetonitrile) is then applied to the column to elute bound peptides by centrifugation.
eptide Desalting Spin Columns are optimized for desalting 5 µg to 5 mg of peptide sample from protein digests.
Additional materials required
• Triuoroacetic acid (TFA) (Product No. 28904)
Acetonitrile (ACN), LC-MS Grade (Product No. 51101)
•
• Water, LC-MS Grade (Product No. 51140)
• Methanol (MeOH), Optima™ LC-MS Grade (Fisher Scientic, Product No. A456-1)
• Thermo Scientic™Pierce™ Low Protein Binding Microcentrifuge Tubes, 2.0 mL (Product No. 88379 or 88380)
• Microcentrifuge with adjustable rotor speed up to 7,000 × g
• Vacuum centrifuge
Additional information
• Do not exceed the recommended centrifugation speeds as this may damage the column frit. Damaged frits may result in resin leakage, leading
to sample loss and/or damage to the LC system.
• Use low protein-binding microcentrifuge tubes to ensure maximum sample recovery.
• Avoid sample contamination and direct skin contact with solvents and chemicals. Always wear gloves when handling the spin columns and
samples.
For Research Use Only. Not for use in diagnostic procedures.
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Prepare materials
Conditioning/Washing Solution (0.1% TFA in water) — Each sample requires 1.5 mL for processing. Add 2 µL of triuoroacetic acid to
1.
2 mL water.
Elution Solution (0.1% TFA, 50% ACN) — Each sample requires 600 µL for elution. Add 2 µL of triuoroacetic acid to 1 mL water and
2.
1 mL acetonitrile. Mix well.
(Optional) TMT Washing Solution (5% MeOH, 0.1% TFA in water) — Each sample will require 300 µL of this solution for processing. Add 50
3.
µL of methanol and 1 µL of triuoroacetic acid to 950 µL of water and mix well.
ean up samples
Cl
Condition the spin columns
Remove the white tip from the boom of the column and discard. Place the column into a 2 mL microcentrifuge tube.
1.
Centrifuge at 5,000 × g for 1 minute to remove the solution and pack the resin material. Discard the liquid.
2.
Note: Do not exceed recommended centrifugation speeds.
Remove the top screw cap and load 300 µL of ACN into the column. Replace the cap, place the spin column back into a 2 mL microcentrifuge
3.
tube and centrifuge at 5,000 × g for 1 minute. Discard ACN and repeat the wash step.
Wash the spin column twice with 0.1% TFA in water, as described in step 3. The column is now conditioned and ready for use.
4.
Bind and wash samples
Dissolve 5-5,000 µg of digested sample in 300 µL of 0.1% TFA in water.
1.
Note: Peptide samples need to be completely dissolved and free of organic solvent (e.g., ACN, DMSO, etc.).
Place the spin column into a 2 mL microcentrifuge tube. Load 300 µL of the sample solution onto the column, replace the top cap and
2.
centrifuge at 3,000 × g for 1 minute. Discard the owthrough.
Place the column into a 2 mL microcentrifuge tube. Load 300 µL of 0.1% TF
3.
to collect the wash. Discard the eluate and repeat this step 2 more times for a total of 3 washes.
Note: TMT-labeled samples require 2 additional column washes with 300 µL of 5% MeOH, 0.1% TFA solution to completely remove the excess
TMT reagent.
A solution onto the column and centrifuge at 3,000 × g for 1 minute
Elute desalted peptides
Place the column into a new 2 mL microcentrifuge tube. Load 300 µL of the 50% ACN, 0.1% TFA solution and centrifuge at 3,000 × g for 1
1.
minute to collect the eluate.
Apply another 300 µL of the 50% ACN, 0.1% TFA solution and centrifuge at 3,000 × g for 1 minute, collecting the eluate into the same
2.
microcentrifuge tube.
Evaporate the liquid contents of each sample tube to dryness using vacuum centrifugation (e.g., SpeedVac concentrator).
3.
Re-suspend dry samples in an appropriate volume of 0.1% formic acid (FA) before LC-MS analysis.
4.
(Optional) Determine the peptide concentration and yield with a peptide quantitation assay.
5.
Related products
Product Cat. no.
BCA Protein Assay Kit 23227
Pierce™ Quantit
Pierce™ Quantitative Colorimetric Peptide Assay 23275
TMTsixplex™ Isobaric Label Reagent Set 90061
TMTsixplex™ Isobaric Mass Tagging Kit 90064
TMT10plex™ Isobaric Label Reagent Set 90110
TMT10plex™ Isobaric Mass Tagging Kit 90113
iodoTMTsixplex™ Label Reagent Set 90101
iodoTMTsixplex™ Isobaric Mass Tag Labeling Kit 90103
aminoxyTMTsixplex™ Label Reagent Set 90401
Pierce™ Mass Spec Sample Prep Kit for Cultured Cells 84840
Pierce™ HeLa Digest Protein Standard 88328
Pierce™ Trypsin Protease, MS Grade 90057
Trifluoroacetic Acid, Sequencing Grade 28904
Water, LC-MS Grade 51140
Acetonitrile (ACN), LC-MS Grade 51101
ative Fluorometric Peptide Assay 23290
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Pierce™ Peptide Desalting Spin Columns
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Troubleshooting
Observation Possible cause Recommended action
Low peptide yields. Low protein yield following lysis and
Unsuccessful recovery. Incorrect centrifuge speeds were
Low peptide/protein indentification
numbers.
the protein extraction procedure.
Lyophilized/dried peptide samples
were not completely solubilized
before sample loading onto the spin
column.
Organic solvents were not completely
removed before loading sample onto
the column.
Samples were not properly acidified
prior to loading onto the column.
used.
Dried, cleaned peptide samples are
not completely recovered from the
sample tube.
Sample load was too low (<5 µg). Estimate peptide concentration using the Thermo Scientific™ Pierce
Incorrect chromatography or mass
spectrometer instrument settings
were used.
Estimate protein concentration using Thermo Scientific™ BCA Protein
Assay Kit (Product No. 23227).
Use an alternative protein extraction procedure.
Increase vortexing/sonication time to completely dissolve the dried
peptide sample.
Ensure the sample is completely dry before resuspending in 0.1% TFA
solution.
Check pH of the sample solution. For the best retention of peptides, pH
should be between 2.5-3.5. Adjust pH if necessary with a dilute formic
acid, acetic acid, or trifluoroacetic acid solution.
Ensure a proper centrifuge speed is used. To convert from revolutions
per minute (rpm) to g, use the following formula:
g
= (1.118 × 10-5) RS2 where g is the relative centrifugal force, R is the
rotor radius in centimeters, and S is the centrifuge speed in rpm. For
example, centrifugation of a sample at 5,000 rpm in a microcentrifuge
having a rotor radius of 7 cm will deliver a centrifugal force of 1,957 ×
Note: Calculated centrifuge speed is only written in the form
of (XXXX × g).
Increase vortexing/sonication time to completely dissolve the dried
peptide sample. For smaller sample loads, repeated aspiration of
dissolving solution using a pipette may improve recovery.
™
Quantitative Fluorometric Peptide Assay (Product No. 23290) or Thermo
Scientific™ Pierce™ Quantitative Colorimetric Peptide Assay Kit (Product
No. 23275).
Use low protein-binding tubes for handling of the samples and fraction
collection.
Consult instrument user manuals or online resources to determine the
optimal instrument settings for your system.
Verify LC-MS system performance with the Thermo Scientific™ Pierce
™
HeLa Digest Protein Standard (Product No. 88328).
g.
Manufacturer: Pier
The information in this guide is subject to change without notice.
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17 August 2017
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