Purification or Removal of Phosphorylated Biomolecules

TiO₂ Mag Sepharose^®

Phosphorylation is a common reversible post-translational modification involved in the regulation of many essential biological processes. Phosphoproteins and phosphopeptides are usually present at very low concentrations and ionize poorly, making their detection by MS difficult.

TiO₂ Mag Sepharose magnetic beads simplify capture and enrichment of phosphopeptides by titanium dioxide (TiO₂)-based chromatography (Figure 5.5). TiO₂ has high affinity for phosphopeptides and provides efficient enrichment of phosphopeptides from complex samples.

Bead characteristics

Characteristics of TiO₂ Mag Sepharose^® beads are shown in Table 5.2.

Purification Example

Two phosphorylated proteins (a-casein and b-casein) and one nonphosphorylated protein (bovine serum albumin) were reduced and alkylated with Tris(2-carboxyethyl) phosphine (TCEP) and iodoacetamide, respectively, followed by trypsin-digestion. A total of 50 pmol of each protein digest was mixed and applied to the magnetic beads. After enrichment, the eluates were lyophilized and dissolved in 20% acetonitrile with 0.1% trifluoroacetic acid (TFA, 20 µl) and analyzed by MALDI-ToF MS.

TiO₂ Mag Sepharose^® detected five peptides, with a ratio of 2.5 between phosphopeptides and nonphosphorylated peptides. Also, after a 100-fold dilution of the eluate, two phophopeptides could still be detected. The experimental conditions and mass spectrograms are shown in Figure 5.6.

Performing a Separation

Sample preparation

For complex samples, such as cell lysate digests, it is recommended to perform a desalting step by use of for example an RPC/C18 cartridge or similar for efficient phosphopeptide enrichment.

Dilute the sample with a minimum of four volumes of binding buffer or dissolve lyophilized sample in binding buffer. Keep sample volumes small, preferably max 100 µl, however up to 250 µl may be used.

Enrichment of phosphorylated proteins

Use the magnetic rack with the magnet in place to attract the beads before each liquid removal step.

1. Prepare the Mag Sepharose beads
   1. Mix the medium slurry throughly by vortexing. Dispense 50 µl of the homogenous medium slurry into an Eppendorf tube.
   2. Place the Eppendorf tube in the magnetic rack to attract the beads.
   3. Remove the storage solution.
2. Equilibration
   1. Add 500 µl binding buffer and resuspend the medium.
   2. Remove the liquid.
3. Apply the sample
   1. Add 50 µl to 250 µl sample.
   2. Resuspend the medium and incubate for 30 min with slow end-over-end mixing or by using a benchtop shaker.
   3. Remove the liquid.
4. Wash 1
   1. Add 500 µl binding buffer and resuspend the medium.
   2. Remove the liquid.
5. Wash 2 and 3 (perform this step twice)
   1. Add 500 µl wash buffer and resuspend the medium.
   2. Remove the liquid.
6. Elution
   1. Add 50 µl elution buffer.
   2. Resuspend the medium and incubate for 5 min.
   3. Remove and collect the eluted fraction. The collected fraction contains the main part of the protein. If needed repeat the elution.

MS analysis

Eluates must be evaporated or neutralized with formic acid or trifluoroacetic acid before analysis with MALDI-ToF. Suitable solvent for evaporated samples is 20% acetonitrile acidified with 0.1% trifluoroacetic acid. For LC-MS analysis using reversed phase chromatography (RPC) the eluates must firstly be evaporated and resuspended in formic acid to a final concentration of 0.1%.