Rapid, single-step procedure for the identification of transglutaminase-mediated isopeptide crosslinks in amino acid digests.
Tissue transglutaminase (tTG) is a calcium-activated enzyme which can covalently crosslink the epsilon-amino group of a peptide-bound lysine into the gamma-carboxamide group of a peptide-bound glutamine, forming a epsilon-(-gamma-glutamyl)lysine isopeptide bond. We have developed a sensitive, single-step method for the isolation and detection of tTG-mediated isopeptide bonds from purified proteins and tissue homogenates. This method offers significantly improved resolution over current techniques, and obviates the need for multi-column systems or costly fluorescence monitors. By using enzymatic proteolysis, derivatization with phenylisothiocyanate, and a simple elution gradient for HPLC, we were able to determine the frequency of crosslinks in purified fibrin (1.7 mol of isodipeptide per mol of fibrin), crosslinked tau proteins (0.75 mol of isodipeptide per mol of tau), and whole-tissue liver homogenates (0.5 nmol of isodipeptide per mg of total protein).
Miller
M L
ML
Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, 35294-0017, USA.
Johnson
G V
GV
D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003432:Cross-Linking Reagents; D004151:Dipeptides / Q000032:analysis*; D017879:Isothiocyanates; D008099:Liver / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D013861:Thiocyanates / Q000737:chemistry; D011503:Transglutaminases / Q000378:metabolism*
D000596:Amino Acids; D003432:Cross-Linking Reagents; D004151:Dipeptides; D017879:Isothiocyanates; D013861:Thiocyanates; C005441:phenylisothiocyanate; C001299:epsilon-(gamma-glutamyl)-lysine; D011503:Transglutaminases