Evidence for phosphorylation of serine 753 in CFTR using a novel metal‐ion affinity resin and matrix‐assisted laser desorption mass spectrometry

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane C1− channel regulated by protein phosphorylation. To identify cAMP‐dependent protein kinase (PKA)‐phosphorylated residues in full‐length CFTR, immobilized metal‐ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron‐loaded (Fe 3+) nitrilotriacetic (NTA) Sepharose compared to iminodiacetic acid (IDA) metal‐chelating matrices was demonstrated using a PKA‐phosphorylated recombinant NBD1‐R protein from CFTR. Fe 3+‐loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly‐His‐containing peptides were co‐purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA‐phosphorylated full‐length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix‐assisted laser desorption mass spectrometry (MALDI/MS) with post‐source decay (PSD) analysis and collision‐induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO 3 and H 3 PO 4 from the parent ions. Peptide sequence and phosphorylation at CFTR residues 660 Ser, 737 Ser, and 795 Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 700 Ser, 712 Ser, 768 Ser, and 813 Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 753 Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 753 Ser in full‐length CFTR.