Impact of limited oxidation on protein ion mobility and structure of importance to footprinting by radical probe mass spectrometry

The effect of hydroxyl radical induced oxidation on the collision cross-sections of hen egg lysozyme and bovine ubiquitin was investigated by travelling wave ion mobility mass spectrometry for the first time. The oxidized ions of lysozyme and ubiquitin share common collision cross-sections with their unoxidized counterparts suggesting that they share common structures that were unaffected by limited oxidation. In the case of bovine ubiquitin, two distinct conformers were detected for the protein in its unoxidized and oxidized states though no change in the levels of each was observed upon oxidation. This supports the validity of Radical Probe Mass Spectrometry (RP-MS) using an electrical discharge source for protein footprinting experiments. Travelling wave ion mobility mass spectrometry has been used for the first time to confirm that limited oxidation does not have an impact on the global structure of proteins.     

Future directions of structural mass spectrometry using hydroxyl radical footprinting

Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein structure and dynamics. It has been successfully applied in the analysis of protein structure, protein folding, protein dynamics, and protein–protein and protein–DNA interactions. Using synchrotron radiolysis, exposure of proteins to a ‘white’ X‐ray beam for milliseconds provides sufficient oxidative modification to surface amino acid side chains, which can be easily detected and quantified by mass spectrometry. Thus, conformational changes in proteins or protein complexes can be examined using a time‐resolved approach, which would be a valuable method for the study of macromolecular dynamics. In this review, we describe a new application of hydroxyl radical protein footprinting to probe the time evolution of the calcium‐dependent conformational changes of gelsolin on the millisecond timescale. The data suggest a cooperative transition as multiple sites in different molecular subdomains have similar rates of conformational change. These findings demonstrate that time‐resolved protein footprinting is suitable for studies of protein dynamics that occur over periods ranging from milliseconds to seconds. In this review, we also show how the structural resolution and sensitivity of the technology can be improved as well. The hydroxyl radical varies in its reactivity to different side chains by over two orders of magnitude, thus oxidation of amino acid side chains of lower reactivity are more rarely observed in such experiments. Here we demonstrate that the selected reaction monitoring (SRM)‐based method can be utilized for quantification of oxidized species, improving the signal‐to‐noise ratio. This expansion of the set of oxidized residues of lower reactivity will improve the overall structural resolution of the technique. This approach is also suggested as a basis for developing hypothesis‐driven structural mass spectrometry experiments. Copyright © 2010 John Wiley & Sons, Ltd.     

On‐surface Fenton and Fenton‐like reactions appraised by paper spray ionization mass spectrometry

On‐surface degradation of sildenafil (an adequate substrate as it contains assorted functional groups in its structure) promoted by the Fenton (Fe²⁺/H₂O₂) and Fenton‐like (Mⁿ⁺/H₂O₂; Mⁿ⁺ = Fe³⁺, Co²⁺, Cu²⁺, Mn²⁺) systems was investigated by using paper spray ionization mass spectrometry (PS‐MS). The performance of each system was compared by measuring the ratio between the relative intensities of the ions of m/z 475 (protonated sildenafil) and m/z 235 (protonated lidocaine, used as a convenient internal standard and added to the paper just before the PS‐MS analyzes). The results indicated the following order in the rates of such reactions: Fe²⁺/H₂O₂ ≫ H₂O₂ ≫ Cu²⁺/H₂O₂ > Mⁿ⁺/H₂O₂ (Mⁿ⁺ = Fe³⁺, Co²⁺, Mn²⁺) ~ Mⁿ⁺ (Mⁿ⁺ = Fe²⁺, Fe³⁺, Co²⁺, Cu²⁺, Mn²). The superior capability of Fe²⁺/H₂O₂ in causing the degradation of sildenafil indicates that Fe²⁺ efficiently decomposes H₂O₂ to yield hydroxyl radicals, quite reactive species that cause the substrate oxidation. The results also indicate that H₂O₂ can spontaneously decompose likely to yield hydroxyl radicals, although in a much smaller extension than the Fenton system. This effect, however, is strongly inhibited by the presence of the other cations, ie, Fe³⁺, Co²⁺, Cu²⁺, and Mn²⁺. A unique oxidation by‐product was detected in the reaction between Fe²⁺/H₂O₂ with sildenafil, and a possible structure for it was proposed based on the MS/MS data. The on‐surface reaction of other substrates (trimethoprim and tamoxifen) with the Fenton system was also investigated. In conclusion, PS‐MS shows to be a convenient platform to promptly monitor on‐surface oxidation reactions.     

Charge‐derivatized amino acids facilitate model studies on protein side‐chain modifications by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

The α‐amino groups of histidine and lysine were derivatized with p‐carboxylbenzyltriphenylphosphonium to form the pseudo dipeptides, PHis and PLys, which can be sensitively detected by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) due to the fixed positive charge of the phosphonium group. Detection limits of PHis and PLys by MALDI‐TOFMS were both 30 fmol with a signal‐to‐noise ratio of 5:1. These pseudo dipeptides were excellent surrogates for His‐ or Lys‐containing peptides in model reactions mimicking proteins with reactive electrophiles, prominently those generated by peroxidation of polyunsaturated fatty acids including 4‐hydroxy‐2(E)‐nonenal (HNE), 4‐oxo‐2(E)‐nonenal (ONE), 2(E)‐octenal, and 2(E)‐heptenal. An air‐saturated solution of linoleic acid (d₀:d₅ = 1:1) was incubated in the presence of Fe(II) and ascorbate with these two pseudo dipeptides, and the reaction products were characterized by MALDI‐TOFMS and liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS). By using PHis and PLys, the previously reported ONE‐derived His‐furan adduct was detected along with evidence for a cyclic α,β‐unsaturated ketone. A dimer formed from ONE was found to react with PHis through Michael addition. Alkenals were found to form two novel adducts with PLys. 2(E)‐Octenoic acid–His Michael adduct and N^ε‐pentanoyllysine were identified as potential protein side‐chain adducts modified by products of linoleic acid peroxidation. In addition, when PHis or PLys and AcHis or BocLys were exposed to the linoleic acid peroxidation, an epoxy‐keto‐ocatadecenoic acid mediated His–His cross‐link was detected, along with the observation of a His–ONE/9,12‐dioxo‐10‐dodecenoic acid–Lys derived pyrrole cross‐link. Copyright © 2009 John Wiley & Sons, Ltd.     

Real‐time reaction monitoring by probe electrospray ionization mass spectrometry

Probe electrospray ionization (PESI) is a modified version of the electrospray ionization (ESI), where the capillary for sampling and spraying is replaced by a solid needle. High tolerance to salts and direct ambient sampling are major advantages of PESI compared with conventional ESI. In this study, PESI‐MS was used to monitor some biological and chemical reactions in real‐time, such as acid‐induced protein denaturation, hydrogen/deuterium exchange (HDX) of peptides, and Schiff base formation. By using PESI‐MS, time‐resolved mass spectra and ion chromatograms can be obtained reproducibly. Real‐time PESI‐MS monitoring can give direct and detailed information on each chemical species taking part in reactions, and this is valuable for a better understanding of the whole reaction process and for the optimization of reaction parameters. PESI‐MS can be considered as a potential tool for real‐time reaction monitoring due to its simplicity in instrumental setup, direct sampling with minimum sample preparation and low sample consumption. Copyright © 2010 John Wiley & Sons, Ltd.     

Ability of N‐acetylcarnosine to protect lens crystallins from oxidation and oxidative damage by radical probe mass spectrometry (RP‐MS)

The application of Radical Probe Mass Spectrometry based on protein footprinting studies is described to investigate the effectiveness of the antioxidant N‐acetylcarnosine (NAC) in preventing oxidative damage to lens crystallins present in the eye of mammals. Despite separate clinical trials which have reported the benefit of administering NAC to the eye as a 1% topical solution for the treatment of human cataract, no evidence was found to suggest that the antioxidant had any significant direct effect on reducing the levels of oxidation within the most abundant lens crystallins, α and β‐crystallin, at the molecular level at increasing concentrations of NAC. The results of this laboratory study suggest that the therapeutic benefit demonstrated in clinical trials is associated with the nature or formulation of the topical solution and/or that the mode of action of NAC as an antioxidant is not a direct one. Copyright © 2010 John Wiley & Sons, Ltd.     

Structural elucidation of N‐oxidized clemastine metabolites by liquid chromatography/tandem mass spectrometry and the use of Cunninghamella elegans to facilitate drug metabolite identification

Cunninghamella elegans is a filamentous fungus that has been shown to biotransform drugs into the same metabolites as mammals. In this paper we describe the use of C. elegans to aid the identification of clemastine metabolites since high concentrations of the metabolites were produced and MSⁿ experiments were facilitated. The combination of liquid chromatography and tandem mass spectrometry with two different ionization techniques and hydrogen/deuterium exchange were used for structural elucidation of the clemastine metabolites. Norclemastine, four isomers of hydroxylated clemastine, and two N‐oxide metabolites were described for the first time in C. elegans incubations. The N‐oxidations were confirmed by hydrogen/deuterium exchange and deoxygenation (?16 Da) upon atmospheric pressure chemical ionization mass spectrometry. By MSⁿ fragmentation it was concluded that two of the hydroxylated metabolites were oxidized on the methylpyrridyl moiety, one on the aromatic ring with the chloro substituent, and one on the aromatic ring without the chlorine. Copyright © 2010 John Wiley & Sons, Ltd.     

Hydroxyl radical probe of the calmodulin-melittin complex interface by electrospray ionization mass spectrometry

The calcium-dependent interaction of calmodulin and melittin is studied through the application of a radical probe approach in which solutions of the protein and peptide and protein alone are subjected to high fluxes of hydroxyl and other oxygen radicals on millisecond timescales. These radicals are generated by an electrical discharge within an electrospray ion source of a mass spectrometer. Condensation of the electrosprayed droplets followed by proteolytic digestion of both calmodulin and melittin has identified residues in both which participate in the interaction and/or are shielded from solvent within the protein complex. Consistent with other theoretical models and available experimental data, the tryptophan residue of melittin at position 19 is shown to be critical to the formation of the complex with the C-terminal domain of peptide enveloped by and protected from oxidation upon binding to the protein. Furthermore, the N-terminal domain (to residue 36) and tyrosine at position 99 in calmodulin are significantly protected from limited oxidation upon the binding of melittin while exposing the phenylalanine residue at position 92 of the flexible loop domain. The N-terminus (through residue 36) of calmodulin is shown to lie in closer proximity to the melittin helix than its C-terminal counterpart (residues 127-148) based upon the protection levels measured at reactive residues within these segments of the protein.     

On‐line solid‐phase extraction for liquid chromatography–mass spectrometry analysis of pesticides

Public concern about pesticides in food and water has increased dramatically in the last two decades. In order to guarantee consumers’ health and safety, analytical methods that could provide fast and reliable answers without compromising accuracy and precision are required. Sample treatment is probably the most tedious and time‐consuming step in many analytical procedures and, despite the significant advances in chromatographic separations and mass spectrometry techniques, sample treatment is still one of the most important parts of the analytical process for achieving good analytical results. Therefore, over the last years, considerable efforts have been made to simplify the stage and to develop fast, accurate, and robust methods that allow the determination of a wide range of pesticides without compromising the integrity of the extraction process. This review article intends to give a short overview of recently developed on‐line solid‐phase extraction, preconcentration, and clean‐up procedures for the determination of pesticides in complex matrices by liquid chromatography–mass spectrometry techniques.     

Initiation of free‐radical polymerization by peroxypivalates studied by electrospray ionization mass spectrometry

Initiation by tert‐butyl peroxypivalate (TBPP), tert‐amyl peroxypivalate (TAPP), 1,1,3,3‐tetramethylbutyl peroxypivalate (TMBPP), or 1,1,2,2‐tetramethylpropyl peroxypivalate (TMPPP) of radical polymerization of methyl methacrylate in toluene solution at 90 °C was studied via polymer end‐group analysis using electrospray ionization mass spectrometry (ESI‐MS). Conclusive peak assignments allowed for measuring the type and concentration of the fragments that actually initiate macromolecular growth after thermal decomposition of these peroxypivalates. It was found that the pivaloyloxy radical moiety undergoes instantaneous decarboxylation to yield an initiating tert‐butyl radical. The alkoxy radical moiety, on the other hand, may generate, via β‐scission reaction, different types of carbon‐centered radicals (together with a ketone) or may undergo a 1,5‐H‐shift reaction, by which reaction an oxygen‐centered radical is transformed into a carbon‐centered hydroxy radical. This hydrogen shift reaction was found in case of TMBPP. Surprisingly, no evidence for initiating alkoxy radicals could be found, not even in case of initiation by TBPP, where the intermediate tert‐butoxy radical undergoes a rapid chain‐transfer reaction with the solvent toluene. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4266–4275, 2004     

Hydroxyl radical probe of the surface of lysozyme by synchrotron radiolysis and mass spectrometry.

A new approach is reported that combines synchrotron radiolysis and mass spectrometry to probe the surface of proteins. Hydroxyl radicals produced upon the radiolysis of protein solutions with synchrotron light for several milliseconds result in the reaction of amino acid side chains. This results in the formation of stable oxidation products where the level of oxidation at the reactive residues is influenced by the accessibility of their side chains to the bulk solvent. The aromatic and sulfur-containing residues have been found to react preferentially in accord with previous peptide studies. The sites of oxidation have been determined by tandem mass spectrometry. The rate of oxidation at these reactive markers has been measured for each of the proteolytic peptides as a function of exposure time based on the relative proportion of modified and unmodified peptide ions detected by mass spectrometry. Oxidation rates have been found to correlate closely with a theoretical measure of the accessibility of residue side chains to the bulk solvent in the native protein structure. The synchrotron-based approach is able to distinguish the relative accessibility of the tryptophan residue side chains of lysozyme at positions 62 and 123 from each other and all other tryptophan residues based on their rates of oxidation.     

Top‐down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry

The original article to which this Erratum refers was published in Rapid Commun. Mass Spectrom. 2006; 20 : 1932–1938.     

Mass spectrometric investigation of protein alkylation by the RNA footprinting probe kethoxal

The reactivity of the RNA footprinting reagent kethoxal (KT) toward proteins was investigated by electrospray ionization-Fourier transform mass spectrometry. Using standard peptides, KT was shown to selectively modify the guanidino group of arginine side chains at neutral pH, while primary amino groups of lysine and N-terminus were found to be unreactive under these conditions. Gas-phase fragmentation of KT adducts provided evidence for a cyclic 1,2-diol structure. Esterification of the 1,2-diol product was obtained in borate buffer, and its structure was also investigated by tandem mass spectrometry. When model proteins were probed with this RNA footprinting reagent, the adducts proved to be sufficiently stable to allow for the application of different peptide-mapping procedures to identify the location of modified arginines. Probing of proteins under native folding conditions provided modification patterns that very closely matched the structural context of arginines in the global protein structure. A strong correlation was demonstrated between the susceptibility to modification and residue accessibility calculated from the known 3D structure. When the complexes formed by HIV-1 nucleocapsid (NC) protein and RNA stemloops SL2 and SL3 were investigated, KT footprinting provided accurate information regarding the involvement of individual arginines in binding RNA and showed different reactivity according to their mode of interaction.     

Electrospray-assisted modification of proteins: a radical probe of protein structure

A new approach is described to probe the structure of proteins through their reactivity with oxygen-containing radicals. Radical-induced oxidative modification of proteins is achieved within an electrospray ion source using oxygen as a reactive nebulizer gas at high needle voltages. This method facilitates the rapid oxidation of proteins as the molecules emerge from the electrospray needle tip. Electrospray mass spectra of both ubiquitin and lysozyme reveal that over 50% of the protein can be modified under these conditions. The radical-induced oxidative modification of amino acid side chains is correlated with their solvent accessibility to obtain information on a protein's higher-order structure. The oxidation sites in hen lysozyme have been identified by proteolysis of the condensed protein solution and tandem mass spectrometry (MS/MS). Oxidation of tryptophan at positions 62 and 123 occurs exclusively over all other tryptophan residues, consistent with the relative solvent accessibilities of the residue side chains based on the NMR structure of the protein. Radical-induced oxidative modification of cysteine (Cys), methionine (Met), tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), proline (Pro), histidine (His), and leucine (Leu) residues is also reported, providing sufficient reactive markers to span a protein sequence. This facile oxidation process could be applied to investigate the molecular mechanism by which reactive oxygen species interact with a particular protein domain as a means to investigate the onset of certain diseases.