Peptide Purification: An RP-HPLC-based Technique to Extract Peptides from Digested Protein Lysates

Abstract

Source: Cheng, L. C. et al. Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer. J. Vis. Exp. (2018)

In this video, we demonstrate reversed-phase high-performance liquid chromatography to purify peptides from a digested protein lysate mixture. These purified peptides are then lyophilized and stored for downstream applications. Analysis of peptides can help understand the mechanics involved in cellular signaling and cancer.

Protocol

1. Reverse Phase Extraction

1. Start with a pre-digested protein lysate in a glass vial. Filter the sample by using a 15 mL 10 kDa cutoff filter. Centrifuge the sample at 3,500 x g using the swing bucket rotor (or 3,500 x g in a fixed angle rotor) at 15 °C until the retentate volume is less than 250 µL (this takes approximately 45 - 60 min). Collect the flow-through and discard the retentate.
   NOTE: The experiment can be paused here. Freeze the samples at -80 °C for later use.
2. To acidify the sample, add approximately 20 µL of 5% trifluoroacetic acid (TFA) per mL of lysate. Mix them well and measure the sample pH by using pH strips. Adjust pH to 2.5 using 5% TFA.
3. Connect the shorter end of a C-18 column to a vacuum manifold. Set the vacuum between 17 and 34 kPa (or according to the manufacturer’s instructions). Using glass pipettes, wet the column with 3 mL of 100% acetonitrile (ACN). Do not let the column dry.
4. Using glass pipettes, equilibrate the column with 6 mL of 0.1% TFA applied as 2x 3 mL. Load the acidified sample into the column. Do not add more than 3 mL at a time. Adjust the vacuum to target about 1 to 2 drops per second.
5. Using glass pipettes, wash the column with 9 mL of 0.1% TFA applied as 3x 3 mL. Elute the column with 2 mL of 40% ACN, 0.1% TFA. Collect two 2 mL fractions into glass culture tubes. Discard the column.
6. Cover the eluate tubes with parafilm and punch 3-5 holes on the cover using a 20G needle. Freeze the eluate on dry ice for at least 30 min until it completely solidifies.
7. Lyophilize the fractions overnight. On the following day, make sure the samples are completely dry before stopping the lyophilizer. Store the tubes in a 50 mL conical tube with delicate wipes at -80 °C.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ultra-Low Temperature Freezer	Panasonic	MDF-U76V
Freezer -20 °C	VWR	scpmf-2020
Swing rotor bucket	ThermoFisher Scientific	75004377
Vacuum manifold	Restek	26080
Lyophilizer	Labconco	7420020
CentriVap Benchtop Vacuum Concentrator	Labconco	7810010
Amicon Ultra-15 Centrifugal Filter Units	Millipore Sigma	UFC901024
End-over-end rotator	ThermoFisher Scientific	415110Q
Glass culture tubes	Fisher Scientific	14-961-26
Parafilm	Fisher Scientific	13-374-12
20G needle	BD	B305175
Kimwipes	Fisher Scientific	06-666A
Screw cap cryotube	ThermoFisher Scientific	379189
Nunc 15 mL conical tubes	ThermoFisher Scientific	12-565-268
Low protein-binding Eppendorf tubes	Eppendorf	22431081
3 mL syringe	BD	309657
Trifluoracetic Acid (TFA)	Fisher Scientific	PI-28904
Acetonitrile (ACN)	Fisher Scientific	A21-1
MilliQ water			Deionized water used to prepare all solutions and bufferes