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

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane Cl^- 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 (Fe³⁺) nitrilotriacetic (NTA) Sepharose compared to iminodiacetic acid (IDA) metal-chelating matrices was demonstrated using a PKA- phosphorylated recombinant NBD1-R protein from CFTR. Fe³⁺-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 HPO₃ and H₃PO₄ from the parent ions. Peptide sequence and phosphorylation at CFTR residues ⁶⁶⁰Ser, ⁷³⁷Ser, and ⁷⁹⁵Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues ⁷⁰⁰Ser, ⁷¹²Ser, ⁷⁶⁸Ser, and ⁸¹³Ser were deduced from peptide mass, metastable fragment ion formation, and pICA consensus sequences. Peptide sequence and phosphorylation at residue ⁷⁵³Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of ⁷⁵³Ser in full-length CFTR.