Transition Metal−Peptide Binding Studied by Metal-Catalyzed Oxidation Reactions and Mass Spectrometry

Abstract

We have identified conditions that allow metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) to correctly identify binding sites of first-row transition metal ions to model peptides. This work extends the applicability of the MCO/MS method to metals other than Cu(II). When the appropriate reducing agent (ascorbate, 10 mM) and oxidizing agent concentrations (1 mM persulfate, atmospheric O2, or both) are used, metal-bound amino acids can be sufficiently and specifically oxidized for clear identification by MS. The MCO reactions with Mn(II), Fe(II), Co(II), and Ni(II) occur to lesser extents than with Cu(II), but oxidation is still extensive enough to allow easy identification of the metal-bound residues. With the exception of aspartic acid, the known metal-binding amino acids of angiotensin I and bacitracin A are oxidized, while no oxidation is observed at nonbinding residues. Failure to oxidize aspartic acid is likely due to the relatively slow reactivity of its carboxylic acid side chain with reactive oxygen species, suggesting that the current MCO/MS protocol is transparent to such acidic residues. Overall, this study indicates that, just as is possible for Cu(II), the MCO/MS method should be suitable for determining the Mn(II)-, Fe(II)-, Co(II)-, and Ni(II)-binding sites of 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.…”

“…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.…”

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

“…Its small size, high reactivity, and water-like properties makes it an excellent reagent for tagging solvent-accessible surface areas. ⋅OH can be produced by Fenton chemistry [20,21], electrochemical methods [22], corona discharge techniques [23], water radiolysis [24][25][26], and photochemical cleavage of H 2 O 2 [27][28][29]. All 20 amino acids can react with ⋅OH; about half of them generate products (often +16 Da modifications) that are readily detectable by MS [25].…”

Post-translational Modifications and Mass Spectrometry Detection

“…Since the seminal work by Finch et al (209) on the identification of oxidative modifications in serum albumin, in vitro oxidative stress systems have been successfully employed to determine the mechanisms of protein oxidation (210)(211)(212)(213)(214)(215). These contributions are considered fundamental both for identifying oxidative modifications that can be considered signatures of ROS activity in biological samples and for the development of the analytical methods necessary for characterizing protein oxidation in vivo.…”