In the structural and functional analysis of proteins (phosphorylated proteins), it is useful to be able to separate phosphopeptides from unmodified (unphosphorylated) peptides after hydrolysis of the protein. One of the most effective methods available is the use of metal chelating packings containing iron (Fe ions). This experiment was derived from the Laboratory Guide to Protein Purification and Characterization by Houzhu Zhu.

Operation method

Separation of phosphopeptides with iminodiacetic acid (iron chelate) Sepharose 6B

Materials and Instruments

EDTA Ammonium acetate Sodium acetate Acetic acid Ferric chloride Tris-HCl Sepharose 6B columns
SpeedVac Rotary Concentrator

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Materials and equipment

Sepharose 6B column (1x6 cm; ~5 ml), containing iminodiacetic acid (chelated with iron), (theoretical capacity of the column is ~2 umol of phosphoprotein) (Pharmacia Biotech Inc.)

Tris-HCl (50 mmoI/L; pH 7.6), containing EDTA (100 mmol/L) and NaCl (500 mmol/L)

Ferric chloride (50 mmol/L)

Acetic acid (0.1 mol/L)

Sodium acetate (0.1 mol/L, pH 5.0) (i.e., 0.1 mol/L acetic acid adjusted to pH 5.0 with NaOH)

Ammonium acetate (1%; pH 6.3 and pH 8.3)

SpeedVac rotary concentrator (Savant Instruments, Inc.)

EDTA (0.2mol/L;pH8.0)

Operating Procedures

All operations were performed at room temperature.

1) Wash the Sepharose 6B column with 4 volumes (20 ml) of iminodiacetic acid.

2) Wash the column with 4 volumes of 50 mmol/L Tris-HCl (pH 7.6) containing 100 mmol/L EDTA and 500 mmol/L NaCl.

3) Wash the column with 4 volumes of _H2O.

4) Wash the column with 4 volumes of 50 mmol/L ferric chloride to saturate the column with iron.

5) Wash the column with 2 volumes (10 ml) of 0.1 mol/L acetic acid to remove loosely bound iron.

6) Add peptide mixture (dissolved in ~4 ml 0.1mol/L acetic acid) to the column.

7) Wash the column with 2 volumes (10 ml) of 0.1mol/L acetic acid to remove unbound peptides.

8) Wash the column with 2 volumes of 0.1 ml/L sodium acetate (pH 5.0).

9) Wash the column with 3 volumes (15 ml) of 1% ammonium acetate (pH 6.3).

10) Elute the phosphopeptides with 4 volumes of 1% ammonium acetate (pH 8.3).

11) Collect 0.5-1.0-ml samples by division. Combine samples by pH (as measured by dot pH paper). Concentrate in a SpeedVac rotary concentrator and analyze for phosphopeptides.

12) Immediately after elution, wash the column with 4 volumes of 0.2 mol/L EDTA. The chelate-free column can be stored in 0.2mol/L EDTA (pH 8.0) at room temperature and reused several times.

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