A STAGE Tip Procedure for Desalting and Purification of Peptides

Published: February 29, 2024

Abstract

This video demonstrates peptide purification through the STAGE Tip procedure. A miniature column containing resin beads bonded to long hydrocarbon chains facilitates hydrophobic interaction-based purification. Upon loading a pre-digested peptide sample into the column, the peptide fragments bind to the resin beads, allowing impurities to pass through. An elution buffer that disrupts the hydrophobic interaction is applied to elute the peptide fragments.

Protocol

1. Purification and Desalting of Peptides Pack three layers of C18 resin in a 200 μl pipette tip for each peptide sample to construct a STop And Go Extraction (STAGE)-tip. Add 1/10 stopping solution (20% acetonitrile [ACN)] and 6% trifluoroacetic acid [TFA]) to samples. Centrifuge samples for pellet precipitation at 13,200 rpm for 10 min. Transfer the supernatant to a new 2 mL tube and discard the pellet. Wash stage tips with 100 μl of 100% AC…

Disclosures

The authors have nothing to disclose.

Materials

15 ml Falcon Tube  VWR 470225-000
25 mL Serological  Fisher Scientific  14955235
Ammonium Bicarbonate (ABC) ThermoFisher Scientific A643-500
Acetic Acid Sigma Aldrich 1099510001
Acetone Sigma Aldrich 34850-1L
Acetonitrile (ACN), MS grade Pierce TS-51101
BSA assay kit  VWR CA62408-940
C18 disks (STAGE tips) Fisher Scientific  13110019
Centrifuge 5810 R Sigma Aldrich EP022628188
Dithiothreitol (DTT) ThermoFisher Scientific R0861 Prepare 1M stock of DTT aliquots and store at  -20°C
Fetal Bovine Serum (FBS) ThermoFisher Scientific  12483020 Thaw out, prepare aliquots and stored at -20°C
HEPES Sigma Aldrich H3375 Prepare 8M Urea/40m HEPES aliquots, store at -20 °C
High-resolution mass spectrometer ThermoFisher Scientific 726042
Iodoacetamide  (IAA) Fisher Scientific  AAA1471514 Prepare 0.55M stock of IAA aliquots and store at  -20°C. Avoid light exposure 
L-glutamine  Fisher Scientific  25030081 Thaw out, prepare aliquots and stored at -20°C
LoBind Microcentrifuge tubes Eppendorf 13-698-794
MaxQuant  https://www.maxquant.org/
PCR strip tubes VWR 76318-802
Penicillin Streptomycin, Amphotericin Sigma Aldrich A5955 Thaw out, prepare aliquots and stored at -20°C
Perseus  https://www.maxquant.org/Perseus/
phosphate-buffered solution (PBS)  Bioshop  PBS408 Store at room temperature 
Probe sonicator  Fisher Scientific  100-132-894
Protease inhibitor cocktail (PIC) tablet  Sigma Aldrich 5892791001 Store at 4°C
RPMI 1640 ThermoFisher Scientific  11875093 Store at 4°C
Sodium Dodecyl Sulphate (SDS) ThermoFisher Scientific  J18220.A1 prepare 2% SDS solution
SpeedVac Centrifuge  Eppendorf 07-748-15
STAGE tipping centrifuge Sonation STC-V2
T75 Flasks ThermoFisher Scientific  156499
Trifluoroacetic acid ThermoFisher Scientific 85183
Tris HCL pH 8.5 Fisher Scientific BP152-1 Store at 4°C
Trypsin/LysC Thermo Fisher Scientific  A40009 aliquot and store at -20°C
Ultrasonic bath VWR 97043-960
Urea  Bioshop  URE001.1

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Cite This Article
A STAGE Tip Procedure for Desalting and Purification of Peptides. J. Vis. Exp. (Pending Publication), e22210, doi: (2024).

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