Identifying the site of spin trapping in proteins by a combination of liquid chromatography, ELISA, and off-line tandem mass spectrometry

Abstract

An off-line mass spectrometry method that combines immuno-spin trapping and chromatographic procedures has been developed for selective detection of the nitrone spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) covalently attached to proteins, an attachment which occurs only subsequent to DMPO trapping of free radicals. In this technique, the protein-DMPO nitrone adducts are digested to peptides with proteolytic agents, peptides from the enzymatic digest are separated by HPLC, and enzyme-linked immunosorbent assays (ELISA) using polyclonal anti-DMPO nitrone antiserum are used to detect the eluted HPLC fractions that contain DMPO nitrone adducts. The fractions showing positive ELISA signals are then concentrated and characterized by tandem mass spectrometry (MS/MS). This method, which constitutes the first liquid chromatography-ELISAmass spectrometry (LC-ELISA-MS)-based strategy for selective identification of DMPO-trapped protein residues in complex peptide mixtures, facilitates location and preparative fractionation of DMPO nitrone adducts for further structural characterization. The strategy is demonstrated for human hemoglobin, horse heart myoglobin and sperm whale myoglobin, three globin proteins known to form DMPO-trappable protein radicals upon treatment with H 2 O 2 . The results demonstrate the power of the new experimental strategy to select DMPO-labeled peptides and identify sites of DMPO covalent attachments.

Measurement of intracellular biomolecular oxidation in liver ischemia–reperfusion injury via immuno-spin trapping

“…The increase in DMPO-protein and -DNA nitrone adducts after I/R injury confirms the protein and DNA radical-scavenging activity of DMPO as previously reported [44][45][46]. The observed decrease in lipid peroxidation after DMPO administration is also in agreement with previous reports showing that DMPO can scavenge lipid radicals [47,48].…”

Oil-Frozen W1/O/W2 Double Emulsions for Dermal Biomacromolecular Delivery Containing Ethanol as Chemical Penetration Enhancer

“…61 In Figure 6, it is observed that ethanol presence in the DE formulation did not diminish the ability of BSA to be "recognized" by the antibody, showing that the BSA-antibody complex formation was comparable for all aqueous samples collected from oil-thawed DEs. This result is not surprising because ethanol is not expected to cause strong denaturation of protein, given that the overall ethanol concentration in the aqueous phase was low.…”

Immuno-spin trapping from biochemistry to medicine: Advances, challenges, and pitfalls. Focus on protein-centered radicals

“…Since the production of the anti-DMPO antibody [23], a number of protein radicals have been detected by DMPO-based IST [21, 32], including myoglobin [23, 34–36], hemoglobin [22, 35, 37–38], cytochrome c [39], neuroglobin [40], Cu, Zn-superoxide dismutase (SOD1) [41–43] and Mn-SOD [44], catalase [33], myeloperoxidase [45–46], eosinophil peroxidase [47], lactoperoxidase [48–49], microglobulin [50], albumin [43, 51], thyroid peroxidase (TPO)[25], α-lactalbumin [52] and Ras GTPase [53]. Oxidants used to generate these radicals included H 2 O 2 , peroxynitrite, Cu 2+ /H 2 O 2 , hypochlorous acid, tert -butylhydroperoxide, peroxymonocarbonate, and reactive species generated from ketoprofen plus UV-A, riboflavin plus UV-A irradiation and nano-TiO 2 plus UV-A irradiation.…”

“…The site of DMPO trapping on hoMb was determined to be Tyr 103 by MS/MS and site-specific mutagenicity experiments [34, 55]. The LC-ELISA-MS-based strategy [36] is a powerful approach to identifying the sites of DMPO reaction with specific radicalized amino acid residues in the model hemoproteins sperm whale Mb, horse heart Mb and human oxyHb. Importantly, this study by Lardinois et [36] showed that peptides containing Tyr-DMPO adducts are better preserved under neutral or basic conditions.…”

“…The LC-ELISA-MS-based strategy [36] is a powerful approach to identifying the sites of DMPO reaction with specific radicalized amino acid residues in the model hemoproteins sperm whale Mb, horse heart Mb and human oxyHb. Importantly, this study by Lardinois et [36] showed that peptides containing Tyr-DMPO adducts are better preserved under neutral or basic conditions. In fact, if a DMPO-protein nitrone adduct is not found, it does not mean that the protein was not radicalized, but that the reaction of DMPO with the radical site may have been slower than the reaction of the radical by other pathways (See competing reaction in Fig.…”