Characterization of glutamine deamidation in a long, repetitive protein polymer via bioconjugate capillary electrophoresis.
We describe a novel method for the determination of glutamine deamidation in a long protein polymer via bioconjugate capillary electrophoresis. Since the current best technique for detection of glutamine (or asparagine) deamidation is mass spectrometry, it is practically impossible to precisely detect the degree of deamidation (i.e., how many residues are deamidated in a polypeptide) in a large protein containing a significant number of glutamine (or asparagine) residues, because the mass difference between native and deamidated residues is just 1 atomic mass unit. However, by covalently attaching polydisperse protein polymers (337 residues) to a monodisperse DNA oligomer (22 bases), the degree of glutamine deamidation, which could not be determined accurately by mass spectrometry, was resolved by free-solution capillary electrophoresis. Electrophoretic separations were carried out after different durations of exposure of the protein to a cyanogen bromide cleavage reaction mixture, which is a general treatment for the purpose of removing an oligopeptide affinity purification tag from fusion proteins. For protein polymers with increasing extents of deamidation, the electromotive force of DNA + polypeptide conjugate molecules increases due to the introduced negative charge of deamidated glutamic acid residues, and consequently CE analysis reveals increasing differences in the electrophoretic mobilities of conjugate molecules, which qualitatively shows the degree of deamidation. Peak analysis of the electropherograms enables quantitative determination of the first four deamidations in a protein polymer. A first-order rate constant of 0.018 h(-1) was determined for the deamidation of a single glutamine residue in the protein polymer during the cyanogen bromide cleavage reaction.
Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, USA.
Jong-In
JI
Won
Robert J
RJ
Meagher
Annelise E
AE
Barron
D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D003488:Cyanogen Bromide / Q000737:chemistry; D019075:Electrophoresis, Capillary; D005813:Genes, Synthetic; D005973:Glutamine / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011108:Polymers / Q000032:analysis / Q000138:chemical synthesis*; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000138:chemical synthesis* / Q000235:genetics* / Q000302:isolation & purification; D020449:Repetitive Sequences, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000577:Amides; D011108:Polymers; D011506:Proteins; D005973:Glutamine; D003488:Cyanogen Bromide