Characterization of bovine surfactant proteins B and C by electrospray ionization mass spectrometry.
Bovine surfactant proteins B (SP-B) and C (SP-C) were analyzed by nano-electrospray ionization mass spectrometry (nano-ESI-MS). The observed molecular masses showed discrepancies compared to the calculated molecular masses using the published amino acid sequences. The number of cysteine residues in the published bovine SP-B amino acid sequences also failed to match the observed mass shift upon reduction of the SP-B dimer. To determine the amino acid sequences of two proteins, SP-B was first digested with trypsin and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS), while SP-C was analyzed by MS/MS in its intact form. The amino acid sequence of bovine SP-B determined here matches the observed molecular mass. The sequence is almost identical to the sheep SP-B except for two amino acid residues, consistent with the proximity of the two species. The correct sequence contains seven cysteine residues. Bovine SP-B exists as dimers and all cysteines are oxidized to form disulfide bonds in physiological conditions, which is in agreement with the observed mass shift upon reduction of the SP-B dimer. These cysteine residues are completely conserved across all species indicating their importance for the biological functions of this surfactant protein. The sequence of SP-C determined here also reveals an L to V substitution at its position 22 compared with the published bovine SP-B sequence.
Liu
Suya
S
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada.
Zhao
Lin
L
Manzanares
Dahis
D
Doherty-Kirby
Amanda
A
Zhang
Cunjie
C
Possmayer
Fred
F
Lajoie
Gilles A
GA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D008969:Molecular Sequence Data; D036103:Nanotechnology; D037701:Pulmonary Surfactant-Associated Protein B / Q000737:chemistry*; D037721:Pulmonary Surfactant-Associated Protein C / Q000737:chemistry*; D016415:Sequence Alignment; D012756:Sheep; D013045:Species Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D037701:Pulmonary Surfactant-Associated Protein B; D037721:Pulmonary Surfactant-Associated Protein C