Optimization of immobilized gallium (III) ion affinity chromatography for selective binding and recovery of phosphopeptides from protein digests

Abstract

Although widely used in proteomics research for the selective enrichment of phosphopeptides from protein digests, immobilized metal-ion affinity chromatography (IMAC) often suffers from low specificity and differential recovery of peptides carrying different numbers of phosphate groups. By systematically evaluating and optimizing different loading, washing, and elution conditions, we have developed an efficient and highly selective procedure for the enrichment of phosphopeptides using a commercially available gallium(III)-IMAC column (PhosphoProfile, Sigma). Phosphopeptide enrichment using the reagents supplied with the column is incomplete and biased toward the recovery and/or detection of smaller, singly phosphorylated peptides. In contrast, elution with base (0.4 M ammonium hydroxide) gives efficient and balanced recovery of both singly and multiply phosphorylated peptides, while loading peptides in a strong acidic solution (1% trifluoracetic acid) further increases selectivity toward phosphopeptides, with minimal carryover of nonphosphorylated peptides. 2,5-Dihydroxybenzoic acid, a matrix commonly used when analyzing phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry was also evaluated as an additive in loading and eluting solvents. Elution with 50% acetonitrile containing 20 mg/mL dihydroxybenzoic acid and 1% phosphoric acid gave results similar to those obtained using ammonium hydroxide as the eluent, although the latter showed the highest specificity for phosphorylated peptides.

Keywords: 2,5-dihydroxybenzoic acid; immobilized metal ion affinity chromatography; mass spectrometry; phosphopeptide enrichment; phosphoproteomics; reversible protein phosphorylation.

FIGURE 1

Phosphopeptide purification using Sigma PhosphoProfile Ga(III)-IMAC spin columns. The panels show representative MALDI mass spectra of a combined tryptic digest of bovine α- and β-casein (500 fmol of each protein) (a) before IMAC enrichment, (b) following IMAC enrichment using the reagents supplied with the Sigma PP Kit, (c) after washing the same column with water and eluting a second time with 0.4 M NH₄oH, or (d) after performing IMAC again using the bind/wash buffer supplied with the PP kit and eluting directly with 0.4 M NH₄oH. The reproducibility of these results was confirmed by repeating each procedure at least three times.

FIGURE 2

Effect of different loading solutions on the selective binding of phosphorylated peptides by Ga(III)-IMAC. The panels show representative MALDI mass spectra of tryptic α- and β-casein peptides loaded in (a) 0.1% and (b) 1% acetic acid, (c) 0.1% and (d) 1% formic acid, or (e) 0.1% and (f) 1% trifluoroacetic acid, and eluted in 0.4 M NH₄OH.

FIGURE 3

Effect of different eluents on the recovery of phosphorylated peptides from Ga(III)-IMAC columns. The panels show MALDI mass spectra of tryptic α- and β-casein peptides loaded in 1% trifluoroacetic acid and eluted in (a) the Sigma PP elution buffer containing 10% phosphoric acid, (b) 0.4 M NH₄OH following initial elution of the same column with the PP elution buffer, (c) NH₄HCO₃ (pH 9.0), or (d) 0.4 M NH₄OH (pH 11) without prior elution using the PP buffer.

FIGURE 4

Effect of DHB on the binding of phosphorylated peptides during Ga(III)-IMAC. Panels show representative MALDI mass spectra of the tryptic α- and β-casein peptides loaded in 50% acetonitrile containing 0.1% trifluoroacetic acid and 20 mg/mL DHB and (a) eluted in Sigma PP elution buffer, (b) eluted in NH₄OH after initial elution of the same column with PP buffer, or (c) detected in the flow-through solution after reloading it onto the Ga-IMAC column in 1% trifluoroacetic acid and eluting with 0.4 M NH₄OH. DHB, 2,5-dihydroxybenzoic acid.

FIGURE 5

Effect of DHB on the recovery of phosphorylated peptides from Ga(III)-IMAC columns. Panels show representative MALDI mass spectra of the tryptic α- and β-casein peptides loaded in 1% TFA and eluted in 50% acetonitrile containing (a) 2.5% formic acid, (b) 1% phosphoric acid, (c) 2.5% formic acid and 20 mg/mL DHB, or (d) 1% phosphoric acid and 20 mg/mL DHB. DHB, 2,5-dihydroxybenzoic acid.