Evidence for phosphorylation of serine 753 in CFTR using a novel metal‐ion affinity resin and matrix‐assisted laser desorption mass spectrometry
David C. A. Neville,Christine R. Rozanas,Elmer M. Price,Darren B. Gruis,Alan S. Verkman,Raymond R. Townsend +5 more
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TLDR
The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane C1− channel regulated by protein phosphorylation, which enables the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest.Abstract:
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. 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 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 PKA 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.read more
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Highly Selective Enrichment of Phosphorylated Peptides from Peptide Mixtures Using Titanium Dioxide Microcolumns
TL;DR: This work reports a highly selective enrichment procedure for phosphorylated peptides based on TiO2microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB), and demonstrates that this new procedure was more selective for binding phosphorylation peptides than IMAC using MALDI mass spectrometry.
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Mass Spectrometry in Proteomics
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Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome.
TL;DR: Several methods for enrichment of phosphorylated proteins and peptides are outlined and various options for their identification and quantitation are discussed with special emphasis on mass spectrometry-based techniques.
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Recent advancements in surface-enhanced laser desorption/ionization-time of flight-mass spectrometry.
TL;DR: The overall history and recent advances in surface enhanced laser desorption/ionization‐time of flight‐mass spectrometry (SELDI‐TOF‐MS) technology is reviewed and its application to functional genomics and biomarker discovery is discussed and exemplified by elucidating a biomarker candidate for prostatic carcinoma.
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The ABC protein turned chloride channel whose failure causes cystic fibrosis
TL;DR: New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.
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