Thermo Scientific 89852 User manual

Pierce™Peptide Desalting Spin Columns
Catalog Numbers 89851 and 89852
Doc. Part No. 2162704 Pub. No. MAN0017280 Rev. A.0
Contents
Product Cat. No. Contents Storage
Pierce™Peptide Desalting Spin Columns
89852 25 spin columns containing 20 mg of resin in a
1:1 water/DMSO slurry Store at 2-8°C.
89851 50 spin columns containing 20 mg of resin in a
1:1 water/DMSO slurry
Product description
The Thermo Scientic™ Pierce™ Peptide Desalting Spin Columns are optimized for desalting 5 µg to 5 mg of peptide sample from protein digests.
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 ecient 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 acidied water by low-speed centrifugation. A
solution with acidied organic solvent (0.1% TFA, 50% acetonitrile) is then applied to the column to elute bound peptides by centrifugation.
Additional materials required
•Triuoroacetic 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 Scientic, Product No. A456-1)
• Thermo Scientic™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.
USER GUIDE
For Research Use Only. Not for use in diagnostic procedures.

Prepare materials
1. Conditioning/Washing Solution (0.1% TFA in water) — Each sample requires 1.5 mL for processing. Add 2 µL of triuoroacetic acid to
2 mL water.
2. Elution Solution (0.1% TFA, 50% ACN) — Each sample requires 600 µL for elution. Add 2 µL of triuoroacetic acid to 1 mL water and
1 mL acetonitrile. Mix well.
3. (Optional) TMT Washing Solution (5% MeOH, 0.1% TFA in water) — Each sample will require 300 µL of this solution for processing. Add 50
µL of methanol and 1 µL of triuoroacetic acid to 950 µL of water and mix well.
Clean up samples
Condition the spin columns
1. Remove the white tip from the boom of the column and discard. Place the column into a 2 mL microcentrifuge tube.
2. Centrifuge at 5,000 × g for 1 minute to remove the solution and pack the resin material. Discard the liquid.
Note: Do not exceed recommended centrifugation speeds.
3. 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
tube and centrifuge at 5,000 × g for 1 minute. Discard ACN and repeat the wash step.
4. 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.
Bind and wash samples
1. Dissolve 5-5,000 µg of digested sample in 300 µL of 0.1% TFA in water.
Note: Peptide samples need to be completely dissolved and free of organic solvent (e.g., ACN, DMSO, etc.).
2. 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
centrifuge at 3,000 × g for 1 minute. Discard the owthrough.
3. Place the column into a 2 mL microcentrifuge tube. Load 300 µL of 0.1% TFA solution onto the column and centrifuge at 3,000 × g for 1 minute
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.
Elute desalted peptides
1. 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
minute to collect the eluate.
2. 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
microcentrifuge tube.
3. Evaporate the liquid contents of each sample tube to dryness using vacuum centrifugation (e.g., SpeedVac concentrator).
4. Re-suspend dry samples in an appropriate volume of 0.1% formic acid (FA) before LC-MS analysis.
5. (Optional) Determine the peptide concentration and yield with a peptide quantitation assay.
Related products
Product Cat. no.
BCA Protein Assay Kit 23227
Pierce™Quantitative Fluorometric Peptide Assay 23290
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
2 Pierce
™
Peptide Desalting Spin Columns

Troubleshooting
Observation Possible cause Recommended action
Low peptide yields. Low protein yield following lysis and
the protein extraction procedure.
Estimate protein concentration using Thermo Scientific™BCA Protein
Assay Kit (Product No. 23227).
Use an alternative protein extraction procedure.
Lyophilized/dried peptide samples
were not completely solubilized
before sample loading onto the spin
column.
Increase vortexing/sonication time to completely dissolve the dried
peptide sample.
Organic solvents were not completely
removed before loading sample onto
the column.
Ensure the sample is completely dry before resuspending in 0.1% TFA
solution.
Samples were not properly acidified
prior to loading onto the column.
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.
Unsuccessful recovery. Incorrect centrifuge speeds were
used.
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) RS2where 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 × g.
Note: Calculated centrifuge speed is only written in the form
of (XXXX × g ).
Dried, cleaned peptide samples are
not completely recovered from the
sample tube.
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.
Low peptide/protein indentification
numbers.
Sample load was too low (<5 µg). Estimate peptide concentration using the Thermo Scientific™Pierce™
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.
Incorrect chromatography or mass
spectrometer instrument settings
were used.
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).
Manufacturer: Pierce Biotechnology, Inc. | Thermo Fisher Scientific | 3747 N. Meridian Road | Rockford, Illinois 61101 USA
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE,
MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all
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17 August 2017