Development of a Methodology Based on Metal-Catalyzed Oxidation Reactions and Mass Spectrometry To Determine the Metal Binding Sites in Copper Metalloproteins

Abstract

Efforts have been made to develop a method that uses metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) to identify the binding site of copper in metalloproteins. This method uses MCO reactions to oxidize the amino acids in the metal-binding site and MS to identify the amino acids that have been oxidized. Several reaction conditions, including Cu(II)/ascorbate/O2, Cu(II)/O2/H2O2, and Cu(II)/ascorbate/O2/H2O2, have been tested at varying concentrations to find the optimum conditions for specific oxidation of only the amino acids bound to copper. For small peptides, such as angiotensin I (Agt I) and [Gln11]-amyloid-beta-protein fragment 1-16 (A beta(1-16)), the optimum conditions for specific modification involve the use of Cu(II)/ascorbate/O2. For a larger protein, azurin, the speed and specificity of the MCO reactions are enhanced by the presence of a relatively high concentration of ascorbate (100 mM) and a small concentration of H2O2 (1 mM). Optimized reaction conditions combined with MS/MS and MSn analysis on a quadrupole ion trap mass spectrometer allow the copper-binding sites to be specifically identified. For Agt I and A beta(1-16), the amino acids bound to copper can be identified without any false positives. For azurin, four of the five amino acids bound to copper are identified with one false positive. This false positive, however, corresponds to the oxidation of Met44, which is probably due to its susceptibility to oxidation and its proximity to the only residue not identified (i.e., Gly45). The results altogether suggest that MCO reactions and MS provide a very promising approach for identifying the amino acid residues bound to copper in metalloproteins.

The effect of histidine oxidation on the dissociation patterns of peptide ions

“…For β2m, an equimolar concentration of copper(II) sulfate was added, while azurin and Fe-SOD natively bind Cu and Fe, respectively, so no metal was added. As we have described previously [14,15,18], each of these proteins is selectively oxidized at the amino acids that bind these metals. Detailed MCO reaction conditions for β2m [15], azurin [14], and Fe-SOD [18] can be found in our previous work.…”

“…As we have described previously [14,15,18], each of these proteins is selectively oxidized at the amino acids that bind these metals. Detailed MCO reaction conditions for β2m [15], azurin [14], and Fe-SOD [18] can be found in our previous work. Angiotensin I, angiotensin II, and the prion peptide were oxidized at concentrations between 100 -500 μM.…”

“…All the peptides and proteins were oxidized using metal-catalyzed oxidation (MCO) reactions as described previously [14,15,18]. β2m, azurin, and Fe-SOD were oxidized at protein concentrations of 20 -60 μM in solutions that were buffered with Tris/TrisHCl or MOPS at 25 -100 mM in open microcentrifuge tubes.…”

“…These methods use radicals (e.g. • OH) to modify solvent-exposed [1][2][3][4][5][6][7][8][9][10] or metal-bound amino acids [11][12][13][14][15][16][17][18]. Tandem MS (MS/MS), typically in conjunction with proteolytic digestion, is then used to identify oxidatively modified residues, and information about protein structure is then derived.…”

Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

“…Hydroxyl radical (·OH) is an interesting covalent probe due to its small size and high reactivity. Of the many methods for generating ·OH [13][14][15][16][17][18][19][20], the photolysis of H 2 O 2 by a pulsed UV laser [21][22][23] is particularly convenient. The microsecond ·OH lifetime implies that this photochemical technique is free of oxidation-induced artifacts if the measurements are conducted under single-exposure conditions [21,24,25].…”