A STAGE Tip Procedure for Desalting and Purification of Peptides

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…

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