Cancer is a disease that begins with mutation of critical genes: oncogenes and tumor suppressor genes. Our research on carcinogenic aromatic hydrocarbons indicates that depurinating hydrocarbon-DNA adducts generate oncogenic mutations found in mouse skin papillomas (Proc. Natl. Acad. Sci. USA 92:10422, 1995). These mutations arise by mis-replication of unrepaired apurinic sites derived from the loss of depurinating adducts. This relationship led us to postulate that oxidation of the carcinogenic 4-hydroxy catechol estrogens (CE) of estrone (E 1 ) and estradiol (E 2 ) to catechol estrogen-3,4-quinones (CE-3, 4-Q) results in electrophilic intermediates that covalently bind to DNA to form depurinating adducts. The resultant apurinic sites in critical genes can generate mutations that may initiate various human cancers. The noncarcinogenic 2-hydroxy CE are oxidized to CE-2,3-Q and form only stable DNA adducts. As reported here, the CE-3,4-Q were bound to DNA in vitro to form the depurinating adduct 4-OHE 1 (E 2 )-1(␣,␤)-N7Gua at 59-213 mol͞mol DNA-phosphate whereas the level of stable adducts was 0.1 mol͞mol DNA-phosphate. In female Sprague-Dawley rats treated by intramammillary injection of E 2 -3,4-Q (200 nmol) at four mammary glands, the mammary tissue contained 2.3 mol 4-OHE 2 -1(␣,␤)-N7Gua͞molDNA-phosphate. When 4-OHE 1 (E 2 ) were activated by horseradish peroxidase, lactoperoxidase, or cytochrome P450, 87-440 mol of 4-OHE 1 (E 2 )-1(␣, ␤)-N7Gua was formed. After treatment with 4-OHE 2 , rat mammary tissue contained 1.4 mol of adduct͞mol DNA-phosphate. In each case, the level of stable adducts was negligible. These results, complemented by other data, strongly support the hypothesis that CE-3,4-Q are endogenous tumor initiators.

Footprinting of proteins by hydroxyl radicals generated on the millisecond to minute timescales to probe protein surfaces suffers from the uncertainty that radical reactions cause the protein to unfold, exposing residues that are protected in the native protein. To circumvent this possibility, we developed a method using a 248 nm KrF excimer laser to cleave hydrogen peroxide at low concentrations (15 mM, 0.04%), affording hydroxyl radicals that modify the protein in less than a microsecond. In the presence of a scavenger (20 mM glutamine), the radical lifetimes decrease to ϳ1 microsecond, yet the reaction timescales are sufficient to provide significant oxidation of the protein. These times are arguably faster than supersecondary protein structure can unfold as a result of the modification. The radical formation step takes place in a nanoliter flow cell so that only one laser pulse irradiates each bolus of sample. The oxidation sites are located using standard analytical proteomics, requiring less than a nanomole of protein. We tested the method with apomyoglobin and observed modifications in accord with solvent accessibility data obtained from the crystal structure of holomyoglobin. Additionally, the results indicate that the F-helix is conformationally flexible in apomyoglobin, in accord with NMR results. We also find that the binding pocket is resistant to modifications, indicating that the protein pocket closes in the absence of the heme group-conclusions that cannot be drawn from current structural methods. When developed further, this method may enable the determination of protein-ligand interfaces, affinity constants, folding pathways, and regions of conformational flexibility. , following the introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), has become an important means for the identification of proteins and the analytical tool of choice for proteomics. Given its sensitivity and speed, MS should also allow a more general characterization of activity, conformation, and interactions of proteins. Here we describe the development of an approach using pulsed-laser, hydroxyl-radical footprinting with product analysis by MS for determining solvent accessibility of a protein, and show early results indicating its potential utility for probing protein-ligand interfaces.Although a protein's chemical nature is not significantly altered when it binds with a ligand, one can determine the effects of binding by evaluating changes in solvent accessibility with and without the bound ligand [1]. The use of hydroxyl radicals for this purpose is attractive because they are sufficiently small to probe solvent accessibility and are highly reactive, as has been convincingly demonstrated by successful footprinting of DNA/protein interactions and RNA folding [2]. Others have advanced this idea for proteins, using both continuous and pulsed sources for the radicals and MS as the analytical tool. Chance and coworkers [3] have demonstrated in an extensive set of articles a more ...

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

A new relatlonshlp between Ion mass and effective cyclotron frequency Is derived for Ions stored In a cublc cell and detected by uslng Fourler transform mass spectrometry. An assessment of colllslonal damping on mass measurement error Is also made. I t Is concluded that frequency perturbation by colllslonal damping, whlle predlcted by the model, Is negllglble at sufflclently low pressure. The mass callbration law is tested at a magnetlc fleld of 1.2 T by uslng major fragment Ions of 1,1,1,2-tetrachloroethane. WHh broad band "chirp" excltatlon of Ions, systematic rather than random errors were dlscovered. The magnitude of these systematic errors increased as the number of Ions Qtored In the cell was Increased. However, H Is predlcted from the calibration law that errors will decrease wHh the square of the magnetic field strength.The Fourier transform mass spectrometer (FTMS) (1) is recognized as a potentially useful analytical instrument because it is capable of high mass range and ultrahigh mass resolution. The demonstration of high resolving power with the FTMS, however, has predated by several years the development of methodology for exact mass measurement and elemental composition assignment.Early attempts to produce a mass calibration scheme sufficiently accurate for elemental composition assignments were not successful. For example, Ledford and McIver (2) reported measurement accuracy ranging from 7 ppm to 151 ppm over the mass range m/z 47 to m/z 264 using an ICR mass spectrometer with electrometer detection. The mass errors were systematic, caused by changes in space charge conditions in the analyzer cell as ions were sequentially observed. Comisarow (3) reported mass measurement accuracy ranging from 0.3 ppm to 80 ppm over the mass range m/z 69 to m / i 1166. The relative measurement errors were found to increase systematically with mass.Mass measurement accuracy of the a few parts per million or less (often sufficient for elemental composition assignment) was achieved in more recent studies. Ledford ( 4 ) et al. investigated mass measurement using a parabolic mass calibration law for cubic analyzer cells. Over a 4 m u mass range, errors of 0.8 ppm were typical, while average errors of 2 ppm over an 18 amu mass range were obtained when a three-parameter fit was used. Wanczek and Allemann (5) reported a novel side-band method for measuring the masses of trapped ions. Errors averaging 1.5 ppm were obtained over the mass range m J z 18 to m/z 170 amu.Although these latter methods (4,5) of calibration represent improvements over earlier work ( 2 , 3 ) , they have not proved to be routinely useful for exact mass determination. One reason is that ion space charge in the analyzer cell affects observed frequencies, and this must be accounted for in accurate measurements. Ledford et al. (4) recognized this in their efforts to develop a mass calibration scheme. They demonstrated that frequency shifts associated with changes in space charge were qualitatively similar to those caused by changes in trap voltage. Whit...

Room-temperature ionic liquids are useful as solvents for organic synthesis, electrochemical studies, and separations. We wished to examine whether their high solubalizing power, negligible vapor pressure, and broad liquid temperature range are advantageous if they are used as matrixes for UV-MALDI. Several different ionic matrixes were synthesized and tested, using peptides, proteins, and poly(ethylene glycol) (PEG-2000). All ionic liquids tested have excellent solubilizing properties and vacuum stability compared to other commonly used liquid and solid matrixes. However, they varied widely in their ability to produce analyte gas-phase ions. Certain ionic matrixes, however, produce homogeneous solutions of greater vacuum stability, higher ion peak intensity, and equivalent or lower detection limits than currently used solid matrixes. Clearly, ionic liquids and their more amorphous solid analogues merit further investigation as MALDI matrixes.

Estrogens can have two roles in the induction of cancer: stimulating proliferation of cells by receptor-mediated processes, and generating electrophilic species that can covalently bind to DNA. The latter role is thought to proceed through catechol estrogen metabolites, which can be oxidized to o-quinones that bind to DNA. Four estrogen-deoxyribonucleoside adducts were synthesized by reaction of estrone 3,4-quinone (E1-3,4-Q), 17 beta-estradiol 3,4-quinone (E2-3,4-Q), or estrone 2,3-quinone (E1-2,3-Q) with deoxyguanosine (dG) or deoxyadenosine (dA) in CH3CO2H/H2O (1:1). Reaction of E1-3,4-Q or E2-3,4-Q with dG produced specifically 7-[4-hydroxyestron-1(alpha, beta)-yl]guanine (4-OHE1-1(alpha, beta)-N7Gua) or 7-[4-hydroxyestradiol-1(alpha, beta)-yl]-guanine (4-OHE2-1(alpha, beta)-N7Gua), respectively, in 40% yield, with loss of deoxyribose. These two quinones did not react with dA, deoxycytidine, or thymidine. When E1-2,3-Q was reacted with dG or dA, N2-(2-hydroxyestron-6-yl)deoxyguanosine (2-OHE1-6-N2dG, 10% yield) and N6-(2-hydroxyestron-6-yl)deoxyadenosine (2-OHE1-6-N6dA, 80% yield), respectively, were formed. These adducts provide insight into the type of DNA damage that can be caused by o-quinones of the catechol estrogens. The estrogen 3,4-quinones are expected to produce depurinating guanine adducts that are lost from DNA, generating apurinic sites, whereas the 2,3-quinones would form stable adducts that remain in DNA, unless repaired. The adducts reported here will be used as references in studies to elucidate the structure of estrogen adducts in biological systems.

Fast photochemical oxidation of proteins (FPOP) is a chemical footprinting method whereby exposed amino-acid residues are covalently labeled by oxidation with hydroxyl radicals produced by the photolysis of hydrogen peroxide. Modified residues can be detected by standard trypsin proteolysis followed by LC/MS/MS, providing information about solvent accessibility at the peptide and even the amino-acid level. Like other chemical footprinting techniques, FPOP must ensure only the native conformation is labeled. Although oxidation via hydroxyl radical induces unfolding in proteins on a timescale of milliseconds or longer, FPOP is designed to limit •OH exposure to 1 μs or less by employing a pulsed laser for initiation to produce the radicals and a radical-scavenger to limit their lifetimes. We applied FPOP to three oxidation-sensitive proteins and found that the distribution of modification (oxidation) states is Poisson when a scavenger is present, consistent with a single conformation protein modification model. This model breaks down when a scavenger is not used and/or hydrogen peroxide is not removed following photolysis. The outcome verifies that FPOP occurs on a time scale faster than conformational changes in these proteins.

Proteins adopt different higher-order structures (HOS) to enable their unique biological functions. Understanding the complexities of protein higher-order structures and dynamics requires integrated approaches, where mass spectrometry (MS) is now positioned to play a key role. One of those approaches is protein footprinting. Although the initial demonstration of footprinting was for the HOS determination of protein/nucleic acid binding, the concept was later adapted to MS-based protein HOS analysis, through which different covalent labeling approaches “mark” the solvent accessible surface area (SASA) of proteins to reflect protein HOS. Hydrogen–deuterium exchange (HDX), where deuterium in D2O replaces hydrogen of the backbone amides, is the most common example of footprinting. Its advantage is that the footprint reflects SASA and hydrogen bonding, whereas one drawback is the labeling is reversible. Another example of footprinting is slow irreversible labeling of functional groups on amino acid side chains by targeted reagents with high specificity, probing structural changes at selected sites. A third footprinting approach is by reactions with fast, irreversible labeling species that are highly reactive and footprint broadly several amino acid residue side chains on the time scale of submilliseconds. All of these covalent labeling approaches combine to constitute a problem-solving toolbox that enables mass spectrometry as a valuable tool for HOS elucidation. As there has been a growing need for MS-based protein footprinting in both academia and industry owing to its high throughput capability, prompt availability, and high spatial resolution, we present a summary of the history, descriptions, principles, mechanisms, and applications of these covalent labeling approaches. Moreover, their applications are highlighted according to the biological questions they can answer. This review is intended as a tutorial for MS-based protein HOS elucidation and as a reference for investigators seeking a MS-based tool to address structural questions in protein science.