Studying protein-carbohydrate interactions by amide hydrogen/deuterium exchange mass spectrometry.
Protein-carbohydrate interactions play a significant role in biological processes. Presented here is the novel application of amide hydrogen/deuterium exchange mass spectrometry (amide exchange-MS) to the study of the interaction between a protein and its carbohydrate substrate. The degree of deuterium incorporation into hen egg lysozyme was monitored with and without substrate to verify that a carbohydrate can provide sufficiently stable protection of the amide hydrogen atoms in a protein's backbone from exchange with deuterated solvent. The substrate protected a number of amide hydrogens from exchange, implying that protein-carbohydrate binding systems will be compatible with amide exchange-MS. Endopolygalacturonase-II (EPG-II) from Aspergillus niger, a pectin-degrading enzyme, was chosen as the first carbohydrate-binding system to be extensively studied using quenched amide exchange-MS. Monitoring the changes in deuterium incorporation of EPG-II in the presence and absence of an oligomer of galacturonic acid implied the location of substrate binding. This study demonstrates the ability of amide exchange-MS to investigate protein-carbohydrate interactions.
Complex Carbohydrate Research Center, Department of Chemistry, University of Georgia, 220 Riverbend Road, Athens, GA 30602-4712, USA.
Daniel
D
King
Michelle
M
Lumpkin
Carl
C
Bergmann
Ron
R
Orlando
D000577:Amides; D000818:Animals; D001234:Aspergillus niger / Q000201:enzymology; D001695:Biology / Q000295:instrumentation*; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000032:analysis*; D002645:Chickens; D018360:Crystallography, X-Ray; D003903:Deuterium; D004187:Disaccharides / Q000494:pharmacology; D006603:Hexuronic Acids / Q000494:pharmacology; D006859:Hydrogen; D013058:Mass Spectrometry / Q000379:methods*; D009113:Muramidase / Q000032:analysis / Q000378:metabolism; D009844:Oligosaccharides / Q000494:pharmacology; D010063:Ovum / Q000737:chemistry / Q000201:enzymology; D011096:Polygalacturonase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000378:metabolism
D000577:Amides; D002241:Carbohydrates; D004187:Disaccharides; D006603:Hexuronic Acids; D009844:Oligosaccharides; D011506:Proteins; C007819:galacturonic acid; C012238:chitotetrose; C032438:chitobiose; D006859:Hydrogen; D003903:Deuterium; C400187:endopolygalacturonase II; D011096:Polygalacturonase; D009113:Muramidase