Combined use of ESI-MS and UV diode-array detection for localization of disulfide bonds in proteins: application to an alpha-L-fucosidase of pea.
A simplified strategy is described for the assignment of disulfide bonds in proteins of medium to high molecular mass (10-30 kDa). The method combines the use of high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) and HPLC with UV diode-array detection (HPLC diode array). The denatured protein is subjected to proteolysis and the peptide mixture is divided into three fractions: (i) underivatized peptides, (ii) ethylpyridylated peptides, and (iii) reduced and ethylpyridylated peptides. The three peptide ensembles are then subjected to chromatographic and spectroscopic analysis. A systematic methodology is described to analyze the large amount of data obtained. The method was applied to the localization of disulfide bonds in alpha-L-fucosidase from pea. The two disulfide bonds were located between residues Cys64 and Cys109 and between Cys162 and Cys169, while Cys127 was free.
Codina
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A
Department of Organic Chemistry, University of Barcelona, Barcelona, Spain.
Fernández
I
I
Giralt
E
E
Martínez
I
I
Ludevid
D
D
D000465:Algorithms; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000032:analysis* / Q000737:chemistry; D001330:Electronic Data Processing; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000737:chemistry; D005644:alpha-L-Fucosidase / Q000737:chemistry* / Q000302:isolation & purification
D004220:Disulfides; D005644:alpha-L-Fucosidase; D014357:Trypsin