Matrix-assisted laser desorption/ionization mass spectrometric peptide mapping of the neural cell adhesion protein neurolin purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis or acidic precipitation.
Neurolin is a cell surface protein involved in the neural regeneration and neogenesis of the central nervous system of goldfish. Its theoretical molecular mass, based on the amino acid sequence translated from the cDNA, is 58 kDa, but in SDS-PAGE it shows an apparent MW of 86 kDa. Neurolin is stated to be a glycoprotein and it contains five potential N- and 96 potential O-glycosylation sites. The complete characterization of the primary structure and initial investigations on the postulated glycosylation of neurolin, immunopurified from goldfish brains, are described. The protein was either digested in situ in the sodium dodecyl sulfate polyacrylamide gel matrix or digested after trichloroacetic acid precipitation. Trypsin and endoprotease Glu-C were used as proteases and matrix-assisted laser desorption/ionization mass spectrometry was applied for direct peptide mapping analysis of the proteolytic mixtures. Various sample preparation techniques were performed and the mass spectra were recorded in both positive- and negative-ion modes.
Kussmann
M
M
Department of Molecular Biology, Odense University, Denmark.
Lässing
U
U
Stürmer
C A
CA
Przybylski
M
M
Roepstorff
P
P
D020410:Activated-Leukocyte Cell Adhesion Molecule; D000595:Amino Acid Sequence; D000818:Animals; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry* / Q000302:isolation & purification; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D006031:Glycosylation; D006054:Goldfish; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014238:Trichloroacetic Acid; D014357:Trypsin / Q000378:metabolism
D020410:Activated-Leukocyte Cell Adhesion Molecule; D015816:Cell Adhesion Molecules, Neuronal; D004220:Disulfides; D006023:Glycoproteins; D014238:Trichloroacetic Acid; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin