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Cornell University

Phosphopeptide enrichment by IMAC

General considerations

This method works well for peptides with one or two phosphorylation sites. Peptides with higher degree(s) of phosphorylation do not recover well.

Materials

  • PHOS-Select Iron Affinity Gel (Sigma P9740)
  • SigmaPrep Spin column (Sigma SC1000)
  • Milli-Q Water
  • HPLC Grade Acetonitrile
  • HPLC Grade Glacial Acetic Acid
  • Reagent Grade Ammonium Hydroxide
  • Reagent Grade Hydrochloric Acid (HCl)
  • pH Test Strips
Note: Continual suspension of the IMAC beads are necessary to ensure efficient binding of the phosphopeptides. This can be achieved by gentle mixing with a vortex mixer or by end-to-end agitation on a shaker. Make sure the spin column is sealed properly so it will not leak during mixing or agitation.

Reagents

Sample Acidifying Solution: 1M HCl = 82.5ml concentrated HCl in 1ml water.

Equilibration/Wash Solution: 250mM acetic acid in 30% acetonitrile = 144ml acetic acid in 10ml 30% acetonitrile/water.

Elution Solution: 150mM ammonium hydroxide in 25% acetonitrile = 102ml ammonium hydroxide in 10ml 25% acetonitrile/water.

Note: Prepare fresh elution solution immediately before use with ammonium hydroxide from a tightly sealed bottle that has been stored at 4°C.

Procedure

  • Remove PHOS-Select Gel from freezer and allow to warm to room temperature.
  • If already in solution, adjust sample pH to 2.5-3.0 with 1M HCl, if necessary, and add acetonitrile to ~30% (v/v). If sample is dry, reconstitute in Equilibration/Wash solution and adjust pH to 2.5-3.0 with 1M HCl if necessary.
Note: The total volume of sample to be subjected to IMAC should be between 100 and 400ml.
  • Rinse a spin column by adding 400ml Equilibration/Wash Solution and mixing end over end for 5 minutes. Spin at 10000 rpm for 30 seconds in microcentrifuge and discard liquid.
  • Invert PHOS-Select Gel bottle several times to form a uniform suspension and measure an appropriate amount into a rinsed spin column. This is best accomplished using a pipette tip that has had the bottom trimmed off to allow the gel to enter the tip. The binding capacity of the gel is 1nmole/ml of packed gel.
  • Equilibrate gel by adding 500ml Equilibration/Wash Solution, vortex and spin at 10000 rpm for 30 seconds in microcentrifuge. Do this three times discarding the liquid from the collection vial.
  • Add sample to the equilibrated gel, vortex and mix end over end for a minimum of 1 hour for efficient binding of the phosphopeptides to the gel.
  • Spin at 10000 rpm for 30 seconds in microcentrifuge and discard liquid (unphosphorylated peptide fraction) in collection vial unless needed for further analysis.
  • Wash gel by adding 200ml Equilibration/Wash Solution to the gel, vortex and spin at 10000 rpm for 30 seconds in microcentrifuge. Discard liquid in collection vial or pool with the unphosphorylated peptide fraction if saving for further analysis. Repeat for a total of two washes.
  • Remove residual Equilibration/Wash Solution by adding 500ml water to the gel, vortex and spin at 10000 rpm in microcentrifuge for 30 seconds. Discard liquid in collection vial. Repeat for a total of two rinses.
  • Elute phosphopeptides by adding 200-400ml Elution Solution, vortex and mix end over end for 5 minutes. Spin at 10000 rpm for 30 seconds in microcentrifuge and save the liquid in the collection vial (phosphorylated peptide fraction) for subsequent mass spec analysis. NOTES: Use a fresh collection vial and do not exceed 5 minutes mixing time which could reduce recovery. Acidify the collected fraction with formic acid for immediate analysis or dry in Speed-Vap if analysis will be performed later.