Structural characterization of bisretinoid A2E photocleavage products and implications for age-related macular degeneration.

Fluorescent bisretinoids, such as A2E and all-trans-retinal dimer, form as a by-product of vitamin A cycling in retina and accumulate in retinal pigment epithelial (RPE) cells as lipofuscin pigments. These pigments are implicated in pathological mechanisms involved in several vision-threatening diseases including age-related macular degeneration. Efforts to understand damaging events initiated by these bisretinoids have revealed that photoexcitation of A2E by wavelengths in the visible spectrum leads to singlet oxygen production and photooxidation of A2E. Here we have employed liquid chromatography coupled to electrospray ionization mass spectrometry together with tandem mass spectrometry (MS/MS), to demonstrate that A2E also undergoes photooxidation-induced degradation and we have elucidated the structures of some of the aldehyde-bearing cleavage products. Studies in which A2E was incubated with a singlet oxygen generator yielded results consistent with a mechanism involving bisretinoid photocleavage at sites of singlet molecular oxygen addition. We provide evidence that one of the products released by A2E photodegradation is methylglyoxal, a low molecular weight reactive dicarbonyl with the capacity to form advanced glycation end products. Methylglyoxal is already known to be generated by carbohydrate and lipid oxidation; this is the first report of its production via bisretinoid photocleavage. It is significant that AGE-modified proteins are detected in deposits (drusen) that accumulate below RPE cells in vivo; drusen have been linked to age-related macular degeneration pathogenesis. Whereas various processes play a role in drusen formation, these findings are indicative of a contribution from lipofuscin photooxidation in RPE.

Structure characterization of lipocyclopeptide antibiotics, aspartocins A, B & C, by ESI-MSMS and ESI-nozzle-skimmer-MSMS.

Three lipocyclopeptide antibiotics, aspartocins A (1), B (2), and C (3), were obtained from the aspartocin complex by HPLC separation methodology. Their structures were elucidated using previously published chemical degradation results coupled with spectroscopic studies including ESI-MS, ESI-Nozzle Skimmer-MSMS and NMR. All three aspartocin compounds share the same cyclic decapeptide core of cyclo [Dab2 (Asp1-FA)-Pip3-MeAsp4-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11]. They differ only in the fatty acid side chain moiety (FA) corresponding to (Z)-13-methyltetradec-3-ene-carbonyl, (+,Z)-12-methyltetradec-3-ene-carbonyl and (Z)-12-methyltridec-3-ene-carbonyl for aspartocins A (1), B (2), and C (3), respectively. All of the sequence ions were observed by ESI-MSMS of the doubly charged parent ions. However, a number of the sequence ions observed were of low abundance. To fully sequence the lipocyclopeptide antibiotic structures, these low abundance sequence ions together with complementary sequence ions were confirmed by ESI-Nozzle-Skimmer-MSMS of the singly charged linear peptide parent fragment ions H-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11-Dab2(1+)-Asp1-FA. Cyclization of the aspartocins was demonstrated to occur via the beta-amino group of Dab2 from ions of moderate intensity in the ESI-MSMS spectra. As the fatty acid moieties do not undergo internal fragmentations under the experimental ESI mass spectral conditions used, the 14 Da mass difference between the fatty acid moieties of aspartocins A (1) and B (2) versus aspartocin C (3) was used as an internal mass tag to differentiate fragment ions containing fatty acid moieties and those not containing the fatty acid moieties. The most numerous and abundant fragment ions observed in the tandem mass spectra are due to the cleavage of the tertiary nitrogen amide of the pipecolic acid residue-3 (16 fragment ions) and the proline residue-11 (7 fragment ions). In addition, the neutral loss of ethanimine from alpha,beta-diaminobutyric acid residue 9 was observed for the parent molecular ion and for 7 fragment ions.

Activation loop phosphorylation modulates Bruton's tyrosine kinase (Btk) kinase domain activity.

Bruton's tyrosine kinase (Btk) plays a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. A number of cell signaling studies clearly show that Btk is activated by Lyn, a Src family kinase, through phosphorylation on activation loop tyrosine 551 (Y(551)). However, the detailed molecular mechanism regulating Btk activation remains unclear. In particular, we do not fully understand the correlation of kinase activity with Y(551) phosphorylation, and the role of the noncatalytic domains of Btk in the activation process. Insect cell expressed full-length Btk is enzymatically active, but a truncated version of Btk, composed of only the kinase catalytic domain, is largely inactive. Further characterization of both forms of Btk by mass spectrometry showed partial phosphorylation of Y(551) of the full-length enzyme and none of the truncated kinase domain. To determine whether the lack of activity of the kinase domain was due to the absence of Y(551) phosphorylation, we developed an in vitro method to generate Y(551) monophosphorylated Btk kinase domain fragment using the Src family kinase Lyn. Detailed kinetic analyses demonstrated that the in vitro phosphorylated Btk kinase domain has a similar activity as the full-length enzyme while the unphosphorylated kinase domain has a very low k(cat) and is largely inactive. A divalent magnesium metal dependence study established that Btk requires a second magnesium ion for activity. Furthermore, our analysis revealed significant differences in the second metal-binding site among the kinase domain and the full-length enzyme that likely account for the difference in their catalytic profile. Taken together, our study provides important mechanistic insights into Btk kinase activity and phosphorylation-mediated regulation.

GPC spin column HPLC-ESI-MS methods for screening drugs noncovalently bound to proteins.

Secondary drug screening methods are described for determining the relative degree of non-covalent binding between drug candidates and a protein of therapeutic interest by gel centrifugation chromatography using GPC spin columns for isolating the protein-drug complexes, under native conditions, and reversed-phase HPLC coupled with ESI-MS for highly resolved and sensitive detection of the drug in the complex, under denaturing conditions. The necessary control samples and limitations of this work are fully described. The GPC spin column HPLC-ESI-MS methodology for screening of drugs non-covalently bound to proteins is illustrated for the non-covalent binding of geldanamycin with Hsp90cat protein.

FTICR-MS applications for the structure determination of natural products.

Natural products are a source of unique chemical entities with specific biological activities of great value to the pharmaceutical industry. However, the determination of unknown structures is usually time consuming and often becomes a bottleneck in the effort to develop natural products into effective drugs. The high-performance features of high magnetic field FTMS have greatly alleviated the structural elucidation bottleneck to meet increasingly shorter discovery timelines for drug candidates based on natural products. The high-performance features of high field FTMS include unsurpassed mass measurement accuracy for elemental formula determination, ultra-high mass resolution for component separation, the ability to perform multiple levels of tandem mass spectrometry for structural elucidation, and moderate sensitivity for limited supply of isolates. A number of applications utilizing these properties of FTMS have been reported recently for the structural elucidation of novel natural product structures originating from terrestrial and marine microorganisms. In this review, FTMS methods and their applications for the structural elucidation and characterization of natural products will be reviewed.

Septocylindrins A and B: peptaibols produced by the terrestrial fungus Septocylindrium sp. LL-Z1518.

Two new peptaibols, septocylindrin A (1) and septocylindrin B (2), related to the well-studied membrane-channel-forming peptaibol alamethicin, were obtained from a terrestrial isolate of the fungus Septocylindrium sp. Both 1 and 2 are linear 19-amino acid peptides with a modified phenylalanine C-terminus. Analysis of the HRMS data indicated that they differ only in the 18th residue, where 1 contains Glu and 2 contains Gln. The structures of these two peptaibols were determined by extensive NMR and HRMS analysis. The absolute configurations of amino acids present in 1 were determined using Marfey's methodology. Both compounds were isolated through bioassay-guided fractionation and exhibited significant antibacterial and antifungal activity.

2-(Quinazolin-4-ylamino)-[1,4]benzoquinones as covalent-binding, irreversible inhibitors of the kinase domain of vascular endothelial growth factor receptor-2.

A series of 2-(quinazolin-4-ylamino)-[1,4] benzoquinone derivatives that function as potent covalent-binding, irreversible inhibitors of the kinase domain of vascular endothelial growth factor receptor-2 (VEGFR-2) has been prepared by ceric ammonium nitrate oxidation of substituted (2,5-dimethoxyphenyl)(6,7-disubstituted-quinazolin-4-yl)amines and by displacement of the chlorine atom of substituted 2-chloro-5-(6,7-disubstituted-quinazolin-4-ylamino)-[1,4]benzoquinones with various amines, anilines, phenols, and alcohols. Enzyme studies were conducted in the absence and presence of glutathione and plasma. Several of the compounds inhibit VEGF-stimulated autophosphorylation in intact cells. Kinetic experiments were performed to study the reactivity of selected inhibitors toward glutathione. Reactivities correlated with LUMO energies calculated as averages of those of individual conformers weighted by the Boltzmann distribution. These results and molecular modeling were used to rationalize the biological observations. The compounds behave as non-ATP-competitive inhibitors. Unequivocal evidence, from mass spectral studies, indicates that these inhibitors form a covalent interaction with Cys-1045. One member of this series displays antitumor activity in an in vivo model.

Mechanism of inactivation of beta-lactamases by novel 6-methylidene penems elucidated using electrospray ionization mass spectrometry.

The reactions of 6-methylidene penems 4-7 with beta-lactamases (TEM-1, SHV-1, Amp-C) were characterized by electrospray ionization mass spectrometry (ESI-MS). The kinetics of the reactions were monitored, demonstrating that only one penem molecule reacts to form an acyl-enzyme complex. For penem 5, the ESI-MS/MS spectrum of the hydrolysis product produced in the reaction was identical to the spectrum generated from a synthesized dihydro[1,4]thiazepine 10, confirming the rearrangement of the penem ring system to a seven-membered dihydro[1,4]thiazepine structure. Gas-phase ESI-MS/MS fragmentation data were rationalized due to tautomerization between imine and enamine substructures. ESI-MS/MS analysis of the T-6 trypsin-digested fragments of TEM-1 and SHV-1 demonstrated that the penems were only attached to Ser-70 of these class A beta-lactamases and that the penem ring structures were rearranged to seven-membered dihydro[1,4]thiazepines.

FTMS structure elucidation of natural products: application to muraymycin antibiotics using ESI multi-CHEF SORI-CID FTMS(n), the top-down/bottom-up approach, and HPLC ESI capillary-skimmer CID FTMS.

The molecular formulas for the structures and substructures of muraymycin antibiotics A1 (C52H90N14O19, MW 1214) and B1 (C49H83N11O18, MW 1113) were determined using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The muraymycin A1 and B1 structures were elucidated by utilizing capillary-skimmer fragmentation with up to five stages of mass spectrometry (MS5). Multi-CHEF, a multiple ion isolation method, was used at each stage of MS(n) to isolate a parent ion and up to four reference ions, for exact-mass calibration. The parent ions were fragmented by SORI-CID and the product ions internally calibrated with average absolute mass errors less than 1 ppm at each stage in the fragmentation processes. Using the top-down/bottom-up approach, the molecular formulas for the antibiotics were determined by summing the elemental formulas of the neutral losses, obtained by measuring the mass differences (<500 Da) between the genetically related sequential parent ion masses in the MS(n) spectra, with the unique elemental formula of the lowest parent ion mass (<500 Da). The structures of 12 additional compounds in the muraymycin complex were elucidated using HPLC ESI capillary-skimmer CID FTMS by correlating their fragmentation patterns with those of muraymycins A1 and B1. Sequential neutral losses of an aminosugar, a valine, a uridine, and an ester fatty acid from the muraymycin parent ions provided diagnostic fragments for characterization.

Structures of the muraymycins, novel peptidoglycan biosynthesis inhibitors.

The muraymycins, a family of nucleoside-lipopeptide antibiotics, were purified from the extract of Streptomyces sp. LL-AA896. The antibiotics were purified by chromatographic methods and characterized by NMR spectroscopy, degradation studies, and mass spectrometry. The structures of 19 compounds were established. The muraymycins constitute a new antibiotic family whose core structure contains a glycosylated uronic acid derivative joined by an aminopropane group to a hexahydro-2-imino-4-pyrimidylglycyl residue (epicapreomycidine) containing dipeptide that is further extended by a urea-valine moiety. Members of this family show broad-spectrum in vitro antimicrobial activity against a variety of clinical isolates (MIC 2 to >64 mug/mL). The muraymycins inhibited peptidoglycan biosynthesis. The fatty acid substituent and the presence or absence of the amino sugar play important roles in biological activity. One of the most active compounds, muraymycin A1, demonstrated protection in vivo against Staphylococcus aureus infection in mice (ED50 1.1 mg/kg).

Multiple ion isolation applications in FT-ICR MS: exact-mass MSn internal calibration and purification/interrogation of protein-drug complexes.

Two new applications using multiple ion isolations in the cell of a Fourier transform-ion cyclotron resonance mass spectrometer equipped with an electrospray ionization source are described. A procedure that uses multiple ion isolations of an analyte and calibrants for internal calibration at each stage in a MSn experiment, under high-resolution exact-mass conditions, for structural characterization/elucidation of angiotensin I and rapamycin is illustrated. Fragment ion mass accuracies < 1.0 ppm are demonstrated and routinely achieved. Purification of a mixture is illustrated by isolating multiple charge states of a protein-drug complex from residual protein for further MSn studies to elucidate the site of covalent drug bonding using IRMPD for a mixture of epidermal growth factor receptor (EGFr) protein and EGFr-drug complex. The procedure developed for multiple ion isolations is referred to as multi-CHEF, multiple correlated harmonic excitation fields, in which tailored waveforms are used to notch out multiple mass regions of a spectrum with minimal off-resonance excitation.

Early discovery drug screening using mass spectrometry.

Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometric methods useful for early discovery drug screening are reviewed. All methods described involve studies of non-covalent complexes between biopolymer receptors and small molecule ligands formed in the condensed phase. The complexes can be sprayed intact directly into the gas phase by ESI-MS using gentle experimental conditions. Gas phase screening applications are illustrated for drug ligand candidates non-covalently interacting with peptides, proteins, RNA, and DNA. In the condensed phase, the complexes can be also isolated, denatured and analyzed by ESI-MS to identify the small molecule ligands. Condensed phase drug screening examples are illustrated for the ESI-MS ancillary techniques of affinity chromatography, ultrafiltration, ultracentrifugation, gel permeation chromatography (GPC), reverse phase-high performance liquid chromatography (RP-HPLC) and capillary electrophoretic methods. Solid phase drug screening using MALDI-MS is illustrated for small molecule ligands bound to MALDI affinity probe tips and to beads. Since ESI and MALDI principally produce molecular ions, high throughput screening is achieved by analyzing mass indexed mixtures.

Further evidence that a cell wall precursor [C(55)-MurNAc-(peptide)-GlcNAc] serves as an acceptor in a sorting reaction.

Previous studies suggested that a Gly-containing branch of cell wall precursor [C(55)-MurNAc-(peptide)-GlcNAc], which is often referred to as lipid II, might serve as a nucleophilic acceptor in sortase-catalyzed anchoring of surface proteins in Staphylococcus aureus. To test this hypothesis, we first simplified the procedure for in vitro biosynthesis of Gly-containing lipid II by using branched UDP-MurNAc-hexapeptide isolated from the cytoplasm of Streptomyces spp. Second, we designed a thin-layer chromatography-based assay in which the mobility of branched but not linear lipid II is shifted in the presence of both sortase and LPSTG-containing peptide. These results and those of additional experiments presented in this study further suggest that lipid II indeed serves as a natural substrate in a sorting reaction.