Method to Site-Specifically Identify and Quantitate Carbonyl End Products of Protein Oxidation Using Oxidation-Dependent Element Coded Affinity Tags (O-ECAT) and NanoLiquid Chromatography Fourier Transform Mass Spectrometry

Abstract

Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1, 4, 7, 10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products.After proteolysis, the resulting AOD-tagged peptides are affinity purified, and analyzed by nanoLC-FTICR-MS, which provides high specificity in extracting co-eluting AOD mass pairs with a unique mass difference and affords relative quantitation based on isotopic ratios. Using this methodology, we have mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic and glutamic acids, were 3 found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities towards oxidation independent of amino acid residue. We expect to extend this methodology to study disease-related oxidation systems.

Redox Proteomics in Selected Neurodegenerative Disorders: From Its Infancy to Future Applications

“…A different type of reagent, oxidation-dependent element coded affinity tags (O-ECAT), has been used to enrich for PCO-modified proteins by employing rare earth metals such as Tb or Ho (241). The structure of the O-ECAT tag is shown in Figure 11, whereby the aminooxy group forms an oxime with aldehydes or ketones and the 1,4,7,10-tetraazacyclododecane, N, N¢, N¢¢, N¢¢¢-tetraacetic acid (DOTA) serves as the metal chelator group.…”

“…Due to the different mass shift caused by the metals [i.e., Tb (158.92 Da), Ho (164.93 Da)] in the reagent tags, the relative heights of the doublet pairs that arise in the MS spectrum can be used for assessing relative quantitation levels of oxidized proteins in the different samples. This method was demonstrated in recombinant human serum albumin, whereby a number of oxidation sites were mapped (241). O-ECAT pairs co-elute in the RP separation, making data analysis more straightforward.…”

Oxidation of proteins: Basic principles and perspectives for blood proteomics

“…Antibodies against the metal-chelator moiety allow the affinity selection of derivatised peptides and proteins, and the probe can be loaded with various metals prior to any labelling of carbonyls in order to provide relative quantification information in MS [102].…”

Fluorescent isotope‐coded affinity tag 2: Peptide labeling and affinity capture

“…Also, the tethered biotin fragmented from the peptides sometimes complicated MS/MS spectra interpretation [15,16]. The possible limitations of the use of the ICAT reagent drove the field toward the development of modified versions of isotopically coded labeling tags such as solid-phase isotope tagging [17,18], cleavable ICAT reagents [15,19,20], visible ICATs [21,22], element-coded affinity tags [23,24] and isobaric tags (iTRAQ) [25][26][27], just to list the most important ones.…”