Identification and comparative quantitation of glycation by stable isotope labeling and LC-MS.
Glycation is a common modification of proteins both in vitro and in vivo. To aid identification and comparative quantitation, a method of stable isotope labeling followed by LC-MS analysis was proposed. The samples were reduced using sodium borohydride or sodium borodeuteride. Reduction of the Schiff base between the amine group and the reducing sugars resulted in a molecular weight increase of 2Da using sodium borohydride or a molecular weight increase of 3Da using sodium borodeuteride. The molecular weight difference of 1Da between peptides containing glycated lysine residue reduced using sodium borohydride or sodium borodeuteride was used to identify glycated peptides and to calculate the glycation difference between samples. The method was used to investigate glycation of a recombinant human IgG1 antibody under native and denaturing conditions. The result demonstrated a good correlation between glycation propensity of lysine residues and their solvent exposure levels.
Protein Characterization, Alexion Pharmaceuticals Inc, 352 Knotter Drive, Cheshire, CT CT06410, United States. Electronic address: Liuh@alxn.com.
Hongcheng
H
Liu
Protein Characterization, Alexion Pharmaceuticals Inc, 352 Knotter Drive, Cheshire, CT CT06410, United States.
Gomathinayagam
G
Ponniah
Protein Characterization, Alexion Pharmaceuticals Inc, 352 Knotter Drive, Cheshire, CT CT06410, United States.
Alyssa
A
Neill
Protein Characterization, Alexion Pharmaceuticals Inc, 352 Knotter Drive, Cheshire, CT CT06410, United States.
Rekha
R
Patel
Protein Characterization, Alexion Pharmaceuticals Inc, 352 Knotter Drive, Cheshire, CT CT06410, United States.
Bruce
B
Andrien
D000595:Amino Acid Sequence; D001894:Borohydrides / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry
D001894:Borohydrides; D007074:Immunoglobulin G; D011994:Recombinant Proteins; C025364:sodium borohydride
Antibody; Glycation; Mass spectrometry; Sodium borohydride