title: Proteolysis and mass spectrometric analysis of an integral membrane: aquaporin 0.
abstract: Due to hydrophobicity, structural analysis of integral membrane proteins poses a formidable challenge for current mass spectrometry-based proteomics approaches. Herein, we demonstrate results from optimized sample preparation and enzymatic proteolysis procedures for the complete primary structure determination of a targeted integral membrane protein, lens aquaporin 0 (AQP0). Plasma membrane from bovine lens tissue was alkali treated and tryptic digestion was performed in optimized acetonitrile-ammonium bicarbonate solution. Full sequence coverage of AQP0 was observed as tryptic peptides using both matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and capillary liquid chromatography tandem mass spectrometry (cLC/MS/MS). An amino acid mutation of Thr to Ile/Leu at residue 199 was deduced based on MS/MS results. In a complementary effort to fully sequence the protein, peptic digestion was developed to take advantage of hydrophobic protein solubility in organic acid as well as the decreased activity of pepsin at low pH. Peptic digestion in 10% formic acid (pH 1.2) generated peptides of 500 to 3000 Da and gave 100% sequence coverage by cLC/MS/MS. In addition to post-translational modifications reported previously, a new phosphorylation site at serine 229 and two oxidation sites at tryptophan 202 and 205 were detected on the protein. These methodologies provide complementary detergent- and CNBr-free procedures for detailed analysis of this important membrane channel protein and offer promise for analysis of the integral membrane proteome.
authors.affiliation: Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Han
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Schey
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D005136:Eye Proteins / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
chemical_list: D005136:Eye Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D014357:Trypsin; D010434:Pepsin A