Determination of exposed sulfhydryl groups in heated beta-lactoglobulin A using IAEDANS and mass spectrometry.
This paper takes a new approach to determining which sulfhydryl groups are exposed during the heat denaturation of bovine beta-lactoglobulin A. The sulfhydryl groups exposed after heating were blocked with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS). The results show that IAEDANS is a suitable blocking agent, and its absorbance at 336 nm enabled the quantification of exposed sulfhydryl groups in a mixture of protein species by gel permeation chromatography. Combined with the specific fragmentation of bound IAEDANS by matrix-assisted laser desorption ionization (MALDI) MS/MS in negative ionization mode, this facilitated the identification of peptides that contained blocked cysteines after enzymatic digestion of the protein. During MALDI MS/MS of the peptides, in positive ionization mode, the IAEDANS molecule remained bound to the cysteines, making it possible to identify exactly which cysteine had been exposed after heating. In beta-lactoglobulin A it was found that cysteine 66 and cysteine 160 were predominantly exposed regardless of the length of exposure to heat.
Kehoe
Joseph J
JJ
Moorepark Food Research Centre, Teagasc, Fermoy, County Cork, Ireland.
Brodkorb
André
A
Mollé
Daniel
D
Yokoyama
Emilie
E
Famelart
Marie-Héléne
MH
Bouhallab
Saíd
S
Morris
Edwin R
ER
Croguennec
Thomas
T
D003545:Cysteine / Q000032:analysis; D006358:Hot Temperature; D007782:Lactoglobulins / Q000737:chemistry*; D009282:Naphthalenesulfonates; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013439:Sulfhydryl Reagents
D007782:Lactoglobulins; D009282:Naphthalenesulfonates; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C006421:1,5-I-AEDANS; D003545:Cysteine