Location of disulfide bonds in mature alpha-L-fucosidase from pea.
Fuc-9 is the mature form of a vacuolar alpha-L-fucosidase enzyme which seems to play an important role in plant growth regulation. Fuc-9 is a 202-residue protein containing five Cys residues located at positions 64, 109, 127, 162 and 169. In this study, the disulfide structure of Fuc-9 was determined by MALDI-TOF mass spectrometry (MS), with minimal clean-up of the samples and at a nanomolar scale. Two strategies, based on a specific chemical cleavage (with 2-nitro-5-thiocyanobenzoic acid and alkaline conditions) at the Cys residues and modification of Cys residues by acrylamide/deuterium labeled acrylamide alkylation, were used. Using these methods, the disulfide pairings Cys64-Cys109 and Cys162-Cys169 could be established. The advantages and limitations of our experimental approach are discussed.
Codina
A
A
Department of Organic Chemistry, University of Barcelona, Spain.
Vilaseca
M
M
Tarragó
T
T
Fernández
I
I
Ludevid
D
D
Giralt
E
E
D020106:Acrylamide / Q000378:metabolism; D000478:Alkylation; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism; D005644:alpha-L-Fucosidase / Q000737:chemistry* / Q000378:metabolism
D004220:Disulfides; D010446:Peptide Fragments; D020106:Acrylamide; D005644:alpha-L-Fucosidase; D014357:Trypsin; D003545:Cysteine