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 en codes an apical membrane Cl- channel regulated by protein phosphorylat ion. To identify cAMP-dependent protein kinase (PKA)-phosphorylated re sidues in full-length CFTR, immobilized metal-ion affinity chromatogra phy (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron-loaded (Fe3+) nitrilotriacetic (NTA) Sepharose co mpared to iminodiacetic acid (IDA) metal-chelating matrices was demons trated using PKA-phosphorylated recombinant NBD1-R protein from CFTR. Fe3+-loaded NTA Sepharose preferentially bound phosphopeptides, wherea s acidic and poly-His-containing peptides were co-purified using the c onventional IDA matrices. IMAC using NTA Sepharose enabled the selecti ve recovery of phosphopeptides and identification of phosphorylated re sidues from a complex proteolytic digest. Phosphopeptides from PKA-pho sphorylated full-length CFTR, generated in Hi5 insect cells using a ba culovirus expression system, were purified using NTA Sepharose. Phosph opeptides were identified using matrix-assisted laser desorption mass spectrometry (MALDI/MS) with post-source decay (PSD) analysis and coll ision-induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO3 and H3PO4 f rom the parent ions. Peptide sequence and phosphorylation at CFTR resi dues (660)Ser, (737)Ser, and (795)Ser were confirmed using MALDI/PSD a nalysis. Peptide sequences and phosphorylation at CFTR residues (700)S er, (712)Ser, (768)Ser and (813)Ser were deduced from peptide mass, me tastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue (753)Ser was confirmed using M ALDI/CID analysis. This is the first report of phosphorylation of (753 )Ser in full-length CFTR.