title: Functional probing of arginine residues in proteins using mass spectrometry and an arginine-specific covalent tagging concept.
abstract: The reactivity of arginine residues in model proteins (ubiquitin, cytochrome c, myoglobin, ribonuclease A, lysozyme) was examined using a selective tagging reaction in combination with on-line monitoring of the reaction progress by electrospray ionization mass spectrometry (ESI-MS). The kinetics of this reaction, based on the cyclization of the guanidine group of arginine with 2,3-butanedione and phenylboronic acid at pH 8-10, allow the grouping of arginines in "exposed" or "partially buried" residues, because they differ substantially in their reaction rate constants for the conversion of the guanidine groups. The method allows one to differentiate between different protein conformations as shown for myoglobin and its apo form and native and reduced ribonuclease A: Removal of the heme group in myoglobin resulted in an increased reactivity for the two partially buried arginines. For RNAse A, quantitative reduction of the disulfide bonds lead to the exposure of an additional arginine residue and two different conformations of the reduced protein were observed by ESI-MS that could be distinguished according to their charge-state distribution. Experimentally obtained accessibilities were compared with solvent-accessibility data calculated from 3D structures and substantial agreement between both techniques was observed.
authors: Christian Doppler Laboratory for Molecular Recognition Materials, Institute of Analytical Chemistry and Food Chemistry, University of Vienna, 1090 Vienna, Austria. alexander.leitner@univie.ac.at
authors: Alexander
authors: A
authors: Leitner
authors: Wolfgang
authors: W
authors: Lindner
mesh_terms: D000345:Affinity Labels / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D000345:Affinity Labels; D011506:Proteins; D001120:Arginine