Protein radicals have been detected as catalytic intermediates for an increasing number of enzymes and, as such, play an important role in the proper functioning of biochemical pathways [1
]. Protein radicals, however, are also implicated in oxidative stress and are associated with a wide range of diseases and disorders including cancer, rheumatoid arthritis, atherosclerosis, diabetes mellitus, Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease) and general aging [2
]. Protein radicals, particularly those not intrinsic to a catalytic mechanism, can cause cleavage of the protein backbone, cross-linking of the protein, and the formation of protein peroxy radicals and protein peroxides [3
]. Yet, many aspects of how free radicals are formed, delocalized and propagated within protein structures remain only partly understood.
Protein radical species are generally detected using electron paramagnetic resonance spectroscopy (EPR). Direct EPR spectrometry can be used to detect and identify relatively stable protein-centered free radicals. However, the short lifetimes of these reactive species have generally required either that the samples containing the radical be rapidly frozen after formation [1
] or that the radical be spin trapped to give an adduct more stable than the primary radical [4
]. Nitrone spin traps, of which 5,5-dimethyl-1-pyrroline-N
-oxide (DMPO) is the most commonly employed, often form long-lived adducts that can be seen by EPR for minutes. The lack of resolvable hyperfine coupling constants in the EPR spectra of many protein-derived radicals and/or protein radical adducts, however, makes it difficult to extract useful data regarding the site of radical formation. Consequently, additional experimental strategies such as site-directed mutagenesis and/or amino acid derivatization are often employed in conjunction with EPR to obtain information on the nature of the amino acid-derived radical(s) involved. As previously noted [4
], these experimental strategies suffer from a number of potential artifacts and limitations. Moreover, EPR measurements require significant quantities of protein (100 μM or more) while relying on rather specialized and costly equipment.
Immuno-spin trapping, a technique developed in this laboratory [6
], reduces the amount of protein needed for radical detection experiments from milligram to microgram quantities. In immuno-spin trapping, polyclonal antiserum that recognizes the nitrone spin trap DMPO is used to detect the presence of DMPO covalently attached to proteins. This approach does not involve EPR, but uses the immunology-based methods of enzyme-linked immunosorbent assay (ELISA) and/or Western blotting to measure antigen-antibody binding. Immuno-spin trapping has greatly increased the sensitivity of radical detection and has been used by a number of groups to demonstrate protein radical formation in different proteins exposed to a variety of oxidants [7
]. Nonetheless, the technology does suffer from some of the drawbacks/limitations of spin trapping with EPR detection. In particular, immuno-spin trapping alone cannot be used to unequivocally localize the actual protein residues labeled by the spin trap.
To complement and overcome the limitations of EPR and immuno-spin trapping approaches, methods using mass spectrometry in combination with spin trapping have been developed. Here, the initial protein radicals are trapped with a spin trap molecule to form covalent adducts, and the protein adducts are digested to peptides with proteolytic agents (usually using the hydrolytic enzyme trypsin). The resulting digestion mixture is typically separated by reverse phase chromatography (RP-HPLC) and the eluted peptides analyzed by mass spectrometry (either off-line or on-line). The use of tandem mass spectrometry allows the unequivocal identification of the actual amino acid(s) labeled by the spin trap and has the advantage that only small (picomole or less) amounts of material are required. A series of publications [13
] has illustrated the utility and applicability of this technique in investigating radicals produced by the interaction of various oxidants with myoglobin, hemoglobin, NADH dehydrogenase and cytochrome c peroxidase. In the majority of cases reported [13
], identification of the amino acid trapped by the spin-trapping agent was obtained on-line with tandem mass spectrometry (MS/MS).
When proteins contain only a few sites to which the spin trap is attached, and the spin-trapping efficiency is high, standard LC-MS/MS approaches are generally successful in identifying those sites. However, even when EPR confirms that the spin trap is attached to a particular protein, LC-MS/MS analysis frequently fails to provide information about the sites of spin trap addition. One inherent reason is the low trapping efficiencies of the spin-traps for protein radicals. Consequently, the peptide adducts are generally grossly underrepresented in the general complex peptide mixtures. Thus, to improve the detection of low-abundance peptide adducts from proteolytic digests, selective enrichment methods must be employed prior to reverse phase LC-MS. Herein, we present an approach, which combines chromatographic procedures, immuno-spin trapping, and off-line tandem mass spectrometry for selective detection of protein residues labeled by the spin trap DMPO. This method adds a dimension of selectivity prior to the MS analyses, thereby facilitating the location of DMPO nitrone adducts for the unambiguous structural determination of modified sites in polypeptide chains. The strategy is demonstrated for human hemoglobin, horse heart myoglobin, and sperm whale myoglobin, three globin proteins that are known to form DMPO-trappable protein radicals upon treatment with H2O2.