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Michael L Gross

Publications and source records attributed to Michael L Gross.

At least 37 records · Page 2Linked to original sources
PubMed2004

Electrospray ionization-mass spectrometry and tandem mass spectrometry reveal self-association and metal-ion binding of hydrophobic peptides: a study of the gramicidin dimer.

Gramicidin is a membrane pentadecapeptide that acts as a channel, allowing the passage of monovalent metal ions and assisting in bacterial cell death. The active form is a noncovalently bound dimer. One means to study the self-assembly of this peptide has been to compare the state of the peptide in various solvents ranging from hydrophilic (e.g., trifluoroethanol) to hydrophobic (e.g., n-propanol). In this article, we report the use of electrospray mass spectrometry to study the self-association of gramicidin in various organic and mixed solvents that are introduced directly into the mass spectrometer. The dimer (both homo and hetero) can survive the introduction into the gas phase, and the amount in the gas phase increases with the decreasing dielectric constant of the solvent, reflecting solution-phase behavior. Tandem mass spectrometry data reveal that the stability of dimer in the gas phase decreases with increasing metal ion size, strongly suggesting that the metal ion binds inside the dimer between the monomers.

Dimerization↗
PubMed2004

Synthesis and liquid chromatography/tandem mass spectrometric characterization of the adducts of bisphenol A o-quinone with glutathione and nucleotide monophosphates.

An environmental, estrogen-like substance, bisphenol A (BPA), is the monomer for the production of polycarbonate plastics used in baby bottles, dental sealants, and as a major component of epoxy resin for the lining of food cans. The oxidation of BPA leads to the reactive electrophilic BPA-o-3,4-quinone (BPA-Q), which can damage DNA and may be implicated in cancer initiation. BPA-Q reacts in vitro with 2'-deoxyguanosine 5'-phosphate (dGMP) and 2'-deoxyadenosine 5'-phosphate (dAMP) but not with 2'-deoxycytidine-5'-phosphate and 2'-deoxythymidine 5'-phosphate. In aqueous acetic acid, BPA-Q also reacts with 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) but not with 2'-deoxycytidine and 2'-deoxythymidine. The reactions are accompanied by loss of the modified base (depurination). We determined the structures of the modified bases by primarily tandem mass spectrometry. In mixtures of deoxynuclesides and deoxynucletides treated with BPA-Q, reactions occur more readily with dGMP/dG followed by dAMP/dA. With calf thymus DNA, significant apurinic sites must be produced because we detected the BPA-Q-guanosine adduct in the incubation mixture. We also found that BPA-Q reacts readily with glutathione (GSH) under acidic or neutral conditions, and we characterized the BPA-Q-GSH conjugate with tandem mass spectrometry (MS/MS). The results are consistent with a mechanism of carcinogenesis whereby BPA-Q, formed in vivo and not adequately detoxified by reactions with GSH, reacts with DNA, causing depurination. The adducts reported will also be appropriate references for identification of BPA-Q adducts in environmental and biological systems.

Air Pollutants, Occupational↗
PubMed2004

Doctors in the decent society: torture, ill-treatment and civic duty.

How should physicians act when faced with corporal punishment, such as amputation, or torture? In most cases, the answer is clear: international law, UN resolutions and universal codes of medical ethics absolutely forbid physicians from countenancing torture and corporal punishment in any form. An acute problem arises, however, in decent societies, but not necessarily liberal states, that are, nonetheless, welcome in the world community. The decent society is often governed, in whole or in part, by religious laws, and while these states abridge various human rights they are peace loving, generally tolerant, and offer their citizens wide avenues for political participation. Under these circumstances the prohibition against corporal punishment and torture weakens, often compelling physicians to participate. This is true in two cases. In Rawls' hypothetical nation of Kazanistan, Islamic law is the order of the day, and amputations and corporal punishment play an integral part in the execution of traditional Islamic justice. In Israel, torture is sometimes used to elicit the information needed to thwart impending terror attacks. In each case, a physician's participation is essential. In light of the near universal condemnation that accompanies torture and corporal punishment, physicians can only appeal to norms anchored in collective well-being and concern for life that override respect for human dignity in these societies. Western societies have consistently rejected this reasoning, but it is part and parcel of life in the decent society.

Amputation, Surgical↗
PubMed2003

Probing Ca2+-induced conformational changes in porcine calmodulin by H/D exchange and ESI-MS: effect of cations and ionic strength.

We applied a new method, "protein-ligand interaction using mass spectrometry, titration, and H/D exchange" (PLIMSTEX) [Zhu, M. M. (2003) J. Am. Chem. Soc. 125, 5252-5253], to determine the conformational changes, binding stoichiometry, and binding constants for Ca(2+) interactions with calmodulin (CaM) under varying conditions of electrolyte identity and ionic strength. The outcome shows that CaM becomes less solvent-accessible and more compact upon Ca(2+)-binding, as revealed by the PLIMSTEX curve. The formation of CaM-4Ca species is the biggest contributor to the shape of the titration curve, indicating that the formation of this species accounts for the largest conformational change in the stepwise Ca(2+) binding. The Ca(2+)-binding constants, when comparisons permit, agree with those in the literature within a factor of 3. The binding is influenced by ionic strength and the presence of other cations, although many of these cations do not cause conformational change in apo-CaM. Furthermore, Ca(2+)-saturated CaM exhibits larger protection and higher Ca(2+) affinity in media of low rather than high ionic strength. Both Ca(2+) and Mg(2+) bind to CaM with different affinities, causing different conformational changes. K(+), if it does bind, causes no detectable conformational change, and interactions of Ca(2+) with CaM in the presence of Li(+), Na(+), and K(+) occur with similar affinities and associated changes in solvent accessibility. These metal ion effects point to nonspecific rather than competitive binding of alkali-metal ions. The rates of deuterium uptake by the various CaM-xCa species follow a three-group (fast, intermediate, slow), pseudo-first-order kinetics model. Calcium binding causes the number of amide hydrogens to shift from the fast to the slow group. The results taken together not only provide new insight into CaM but also indicate that both PLIMSTEX and kinetic modeling of H/D exchange data may become general methods for probing protein conformations and quantifying protein-ligand interactions.

Animals↗
PubMed2003

Metabolism and DNA binding studies of 4-hydroxyestradiol and estradiol-3,4-quinone in vitro and in female ACI rat mammary gland in vivo.

Studies of estrogen metabolism, formation of DNA adducts, carcinogenicity, cell transformation and mutagenicity have led to the hypothesis that reaction of certain estrogen metabolites, predominantly catechol estrogen-3,4-quinones, with DNA can generate the critical mutations initiating breast, prostate and other cancers. The endogenous estrogens estrone (E1) and estradiol (E2) are oxidized to catechol estrogens (CE), 2- and 4-hydroxylated estrogens, which can be further oxidized to CE quinones. To determine possible DNA adducts of E1(E2)-3,4-quinones [E1(E2)-3,4-Q], we reported previously that the reaction of E1(E2)-3,4-Q with dG produces the depurinating adduct 4-hydroxyE1(E2)-1-N7Gua [4-OHE1(E2)-1-N7Gua] by 1,4-Michael addition (Stack et al., Chem. Res. Toxicol., 1996, 9, 851). We report here that reaction of E1(E2)-3,4-Q with Ade results in the formation of 4-OHE1(E2)-1-N3Ade by 1,4-Michael addition. The N7Gua and N3Ade depurinating adducts formed both in vitro and in rat mammary gland in vivo were analyzed by HPLC with electrochemical detection and, for some samples, by LC/MS/MS. When E2-3,4-Q was reacted with DNA in vitro, the depurinating adducts 4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua, which are rapidly lost from DNA by cleavage of the glycosyl bond, were formed (>99% of the total adducts), as well as traces of stable adducts, which remain in DNA unless removed by repair. Similar results were obtained when 4-OHE2 was oxidized by horseradish peroxidase, lactoperoxidase, tyrosinase or phenobarbital-induced rat liver microsomes in the presence of DNA. When 4-OHE2 or E2-3,4-Q was injected into the mammary glands of female ACI rats in vivo and the mammary tissue was excised 1 h later, the depurinating adducts 4-OHE2-1-N3Ade and 4-OHE2-1-N7Gua constituted >99% of the total adducts formed. In addition, 4-OHE2 conjugates formed by reaction of E2-3,4-Q with glutathione were also detected. These results demonstrate that the 4-CE are metabolized to CE-3,4-Q, which react with DNA to form primarily depurinating adducts. These adducts can generate the critical mutations that initiate cancer (Chakravarti et al., Oncogene, 2001, 20, 7945; Chakravarti et al., Proc. Am. Assoc. Cancer Res., 2003, 44, 180).

Animals↗
PubMed2003

Quantification of protein-ligand interactions by mass spectrometry, titration, and H/D exchange: PLIMSTEX.

Protein-ligand binding and the concomitant conformational change in the protein are of crucial importance in biophysics and drug design. We report a novel method to quantify protein-ligand interactions in solution by mass spectrometry, titration, and H/D exchange (PLIMSTEX). The approach can determine the conformational change, binding stoichiometry, and affinity in protein-ligand interactions including those that involve small molecules, metal ions, and peptides. Binding constants obtained by PLIMSTEX for four model protein-ligand systems agree with K values measured by conventional methods. At higher protein concentration, the method can be used to determine quickly the binding stoichiometry and possibly the purity of proteins. Taking advantage of concentrating the protein on-column and desalting, we are able to use different concentrations of proteins, buffer systems, salts, and pH in the exchange protocol. High picomole quantities of proteins are sufficient, offering significantly better sensitivity than that of NMR and X-ray crystallography. Automation could make PLIMSTEX a high throughput method for library screening, drug discovery, and proteomics.

Animals↗
PubMed2003

APCs present A beta(k)-derived peptides that are autoantigenic to type B T cells.

Type B T cells recognize peptide provided exogenously but are ignorant of the same epitope derived from intracellular processing. In this study, we demonstrate the existence of type B T cells to an abundant autologous peptide derived from processing of the I-A(k) beta-chain. T cell hybridomas raised against this peptide fail to recognize syngeneic APC despite abundant presentation of the naturally processed epitope but react in a dose-dependent manner to exogenous peptide. Moreover, these hybridomas respond to Abeta(k) peptide extracted from the surface of I-A(k)-expressing APC. This peptide was isolated from B cell lines where it was found in high abundance; it was also present in lines lacking HLA-DM, but in considerably lower amounts. Therefore, type B T cells exist in the naive repertoire to abundant autologous peptides. We discuss the implications of these findings to the potential biological role of type B T cells in immune responses and autoimmune pathology.

Amino Acid Sequence↗
PubMed2003

Specificity of peptide selection by antigen-presenting cells homozygous or heterozygous for expression of class II MHC molecules: The lack of competition.

We isolated and identified naturally processed peptides selected by antigen-presenting cells homozygous for expression of I-A(g7) or I-A(d) class II MHC molecules, or from heterozygous antigen-presenting cells that express I-A(g7) along with I-A(g7PD) or I-A(d). Identification of large numbers of peptides demonstrated that despite being closely related on a structural level, each class II MHC molecule selected for very unique peptides. The large data sets allowed us to definitively establish the preferred peptide-binding motifs critical for selection of peptides by I-A(g7), I-A(g7PD), and I-A(d). Finally, extensive analyses of peptide families reveals that there was little competition among class II MHC alleles for display of peptides and that presence of one allele had minimal impact on the repertoire of peptides selected by another.

Amino Acid Sequence↗
PubMed2003

G-quadruplex formation of thrombin-binding aptamer detected by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry detected the formation of the G-quadruplex structure of the thrombin-binding aptamer, d(GGTTGGTGTGGTTGG), and established its specific interaction with metal ions. One piece of evidence that the bonding in the gas phase is via the G-quadruplex form is the enhanced binding, with respect to other metal ions, of the aptamer with Sr2+, Pb2+, Ba2+, and K+, which are of similar size. Another is the lack of specific binding with controls in which the G's are replaced with A's. The most convincing evidence is the extent of H/D exchange of the gas-phase aptamer as compared to that bound to K+ and Sr2+. The latter two complexes exchange six and nine fewer H's, indicating a significant increase in protection upon binding to the metals. Mass spectrometry will be an important tool in understanding G-quadruplexes, which are particularly important in DNA telomers.

DNA↗
PubMed2003

Structural distinction among inositol phosphate isomers using high-energy and low-energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization.

Electrospray (ESI) collisional-activated dissociation (CAD) tandem mass spectrometric methods for the structural characterization of inositol phosphates (InsPs) using both quadrupole and sector mass spectrometers are described. Under low-energy CAD, the [M + H](+) ions of the positional isomers of inositol phosphates, including inositol mono-, bis- and trisphosphates, yield distinguishable product-ion spectra, which are readily applicable for isomer differentiation. In contrast, the product-ion spectra arising from high-energy CAD (2 keV collision energy, floating at 50%) tandem sector mass spectrometry are less applicable for isomer identification. The differences in the product-ion spectrum profiles among the aforementioned InsP isomers become more substantial and differentiation of positional isomers can be achieved when the collison energy is reduced to 1 keV (floating at 75%). These results demonstrate that the applied collision energies play a pivotal role in the fragmentations upon CAD. The product-ion spectra are similar among the positional isomers of inositol tetrakisphosphates and of inositol pentakisphosphates. Thus, isomeric distinction for these two inositol polyphosphate classes could not be established by the tandem mass spectrometric methods that have achieved such distinctions for the less highly phosphorylated inositol phosphate classes. Under both high- and low-energy CAD, the protonated molecular species of all InsPs undergo similar fragmentation pathways, which are dominated by the consecutive losses of H(2)O, HPO(3) and H(3)PO(4).

Animals↗
PubMed2003

Autoantibodies and CD4 T cells target a beta cell retroviral envelope protein in non-obese diabetic mice.

We determined that, over a biologic time interval, from 4 to 8 weeks of age, female non-obese diabetic (NOD) mice develop antibodies against pancreatic beta-cell-surface antigens depending upon the presence of both the MHC class II susceptibility allele, I-A(g7), and other NOD background genes. We generated a mAb from a pre-diabetic NOD mouse that binds to the surface of insulinoma cells and isolated mouse beta cells, and identified the target as a retroviral envelope glycoprotein expressed on pancreatic beta cells. The cloned and expressed sequence for this protein was recognized by the mAb. The antibody as well as sera from pre-diabetic NOD mice recognized the recombinant protein. Spontaneous T cell reactivity against a peptide from the cloned protein was found in NOD mice. In conclusion, a beta cell retroviral envelope protein is a target antigen that is selected by the NOD mouse immune system early in the pathogenesis of autoimmune diabetes.

Adoptive Transfer↗
PubMed2002

Chemical identification of a low abundance lysozyme peptide family bound to I-Ak histocompatibility molecules.

The processing by antigen-presenting cells (APC) of the protein hen egg-white lysozyme (HEL) results in the selection of a number of peptide families by the class II major histocompatibility complex (MHC) molecule, I-A(k). Some of these families are expressed in very small amounts, in the order of a few picomoles/10(9) APC. We detected these peptides from an extract of class II MHC molecules by using monoclonal anti-peptide antibodies to capture the MHC-bound peptides prior to their examination by HPLC tandem mass spectrometry. Here, we have identified several members of a family of peptides encompassing residues 20-35, which represent less than 1% of the total HEL peptides. Binding analysis indicated that the core segment of the family was represented by residues 24-32 (SLGNWVCAA). Asn-27 (shown in boldface) is the main MHC-binding residue, mapped as interacting with the P4 pocket of the I-A(k) molecule. Analysis of several T cell hybridomas indicated that three residues contacted the T cell receptor: Tyr-23 (P-1), Leu-25 (P3), and Trp-28 (P5). The HEL peptides isolated from the APC extract were sulfated on Tyr-23, but further analysis showed that this modification did not occur physiologically but took place during the peptide isolation.

Alanine↗
PubMed2002

In APCs, the autologous peptides selected by the diabetogenic I-Ag7 molecule are unique and determined by the amino acid changes in the P9 pocket.

We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic I-A(g7) molecule, the P9 pocket of I-A(g7) plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either I-A(g7) molecules or a mutant I-A(g7) molecule in which the beta57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by I-A(g7)-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by I-A(g7) were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified I-A(g7) molecule. These peptides did not favor acidic residues toward the carboxyl terminus.

Adenovirus E2 Proteins↗
PubMed2002

Comparing similar spectra: from similarity index to spectral contrast angle.

We investigated a spectral-contrast-angle (theta) method to determine whether mass spectra of structural isomers are the same or significantly different. This method represents collisionally activated dissociation (CAD) spectra as vectors in space. Mass spectra of different isomers are represented as different vectors, having characteristic lengths and direction. The derived spectral contrast angle, which is a measure of the angle between two vectors corresponding to two closely related spectra, is a measure of whether the mass spectra are the same or significantly different. We compare this method with the similarity index (SI) method and show that the spectral contrast angle method is superior and can differentiate between very similar spectra in cases where the SI cannot. Both methods can be implemented simply in situations where the analyst is called on to decide, on the basis of mass or product-ion spectra, whether reference and unknown compounds are the same or to evaluate the reproducibility of spectra comprised of many peaks.

Chemistry Techniques, Analytical↗
PubMed2002

Multiple-stage mass spectrometric analysis of complex oligosaccharide antibiotics (everninomicins) in a quadrupole ion trap.

Electrospray ionization (ESI) quadrupole ion-trap tandem mass spectrometry (MS/MS) was utilized to characterize a class of complex oligosaccharide antibiotics (everninomicins) that include SCH 27899, everninomicin-D, amino everninomicin (SCH 27900), and SCH 49088 (containing a hydroxylamino-ether sugar). The addition of sodium chloride (approximately 1 microg/mL) facilitates the formation of abundant metal complex ions, and this was used because protonation does not readily occur for most of these compounds. The multiple-stage mass analysis (MS(n)) of the sodiated species provides an important series of fragment ions that are specific for sugar sequence and for some sugar-ring opening. These data suggest a general charge-remote fragmentation pattern with the sodium cation residing in a specific, central location of the sugar chain and fragmentation occurring to trim the end of the molecule. For protonated everninomicin (SCH 27900), however, the proton appears to be mobile during the collisional activation process, opening different fragmentation pathways depending on the proton location. The use of water and acetonitrile with 0.1% acetic acid as the solvent in ESI-MS promotes rapid hydrolysis of the central ortho ester, resulting in the formation of abundant sodiated products that are hydrated. These product ions of the hydrated molecules are likely formed by the same charge-remote fragmentation processes as those that occur for the unhydrolyzed precursor.

Aminoglycosides↗