The disulfide bond pattern of salmon egg lectin 24K from the Chinook salmon Oncorhynchus tshawytscha.
The disulfide bonds in the galactose-specific lectin SEL 24K from the egg of the Chinook salmon Oncorhynchus tshawytscha were determined by mass spectrometry. Four predictive in silico tools were used to determine the oxidation state of cysteines in the sequence and possible location of the disulfide bonds. A combination of tryptic digestion, HPLC separation, and chemical modifications were used to establish the location of seven disulfide bonds and one pair of free cysteines. After proteolysis, peptides containing one or two disulfide bonds were identified by reduction and mass spectral comparison. MALDI mass spectrometry was supported by chemical modification (iodoacetamide) and in silico digestion. The assignments of disulfide bonds were further confirmed by mass spectral fragmentation studies including in-source dissociation (ISD) and collision-induced dissociation (CID). The experimentally determined disulfide bonds and free Cys residues were only partially consistent with those generated by several automated public-domain algorithms.
Yu
Haiqiang
H
Department of Chemistry, University of the Pacific, 3601 Pacific Avenue, Stockton, CA 95211, USA.
Murata
Kenji
K
Hedrick
Jerry L
JL
T Almaraz
Ruben
R
Xiang
Fan
F
Franz
Andreas H
AH
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003553:Cystine / Q000032:analysis; D004220:Disulfides / Q000737:chemistry*; D004527:Egg Proteins / Q000737:chemistry* / Q000302:isolation & purification; D029941:Fish Proteins / Q000737:chemistry* / Q000302:isolation & purification; D037102:Lectins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D012474:Salmon; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D004527:Egg Proteins; D029941:Fish Proteins; D037102:Lectins; D010446:Peptide Fragments; C425164:salmon egg lectin; D003553:Cystine; D014357:Trypsin