A tandem mass spectrometry--based approach is demonstrated for detecting a receptor for Ad37, one of the causative agents for epidemic keratoconjunctivitis. Partial purification of membrane glycoproteins was performed by using lectin-affinity chromatography and SDS-PAGE. Gel bands that were shown to bind Ad37 by using Viral Overlay Protein Blot Assay (VOPBA) were excised, proteolyzed and analyzed by using nanoLC-MS/MS to identify putative receptors contained in a mixture of proteins. Four candidate receptors were identified among approximately 50 proteins based on a search against a protein database. Inhibition of gene delivery mediated by an Ad37 vector, with antibodies against the glycoproteins identified by tandem mass spectrometry, strongly indicated that Membrane Cofactor Protein (MCP), a member of the complement regulatory family of proteins, is the receptor. This rapid and sensitive MS/MS-based strategy is perceived to have wide potential applications for the detection of viral receptors.

Subgroup D adenovirus (Ad) types 8, 19, and 37 (Ad8, -19, and -37, respectively) are causative agents of epidemic keratoconjunctivitis and genital tract infections. Previous studies showed that Ad37 binds to a 50-kDa membrane glycoprotein expressed on human ocular (conjunctival) cells. To identify and characterize the role of the 50-kDa glycoprotein in Ad37 infection, we partially purified this molecule from solubilized Chang C conjunctival cell membranes by using lentil lectin chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liquid chromatography coupled to nano-electrospray ionization-tandem mass spectrometry was subsequently used to identify four Ad37 receptor candidates: CD46, CD87, CD98, and CD147. Immunodepletion analyses demonstrated that the 50-kDa protein is identical to CD46 (also known as membrane cofactor protein). The Ad37, but not Ad5, fiber knob bound to the extracellular domain of CD46, demonstrating a direct interaction of an Ad37 capsid protein with CD46. An antibody specific for the N-terminal 19 amino acids of CD46 also blocked Ad37 infection of human cervical carcinoma and conjunctival cells, indicating a requirement for CD46 in infection. Finally, expression of a 50-kDa isoform of human CD46 in a CD46-null cell line increased cell binding by wild-type Ad37 and gene delivery by an Ad vector pseudotyped with the Ad37 fiber, but not by a vector bearing the Ad5 fiber. Together, these studies demonstrate that CD46 serves as an attachment receptor for Ad37 and shed further light on the cell entry pathway of subgroup D Ads.

Enzymes regulate biological processes through the conversion of specific substrates to products. Therefore, of fundamental interest for every enzyme is the elucidation of its natural substrates. Here, we describe a general strategy for identifying endogenous substrates of enzymes by untargeted liquid chromatography-mass spectrometry (LC-MS) analysis of tissue metabolomes from wild-type and enzyme-inactivated organisms. We use this method to discover several brain lipids regulated by the mammalian enzyme fatty acid amide hydrolase (FAAH) in vivo, including known signaling molecules (e.g., the endogenous cannabinoid anandamide) and a novel family of nervous system-enriched natural products, the taurine-conjugated fatty acids. Remarkably, the relative hydrolytic activity that FAAH exhibited for lipid metabolites in vitro was not predictive of the identity of specific FAAH substrates in vivo. Thus, global metabolite profiling establishes unanticipated connections between the proteome and metabolome that enable assignment of an enzyme's unique biochemical functions in vivo.

Silylation chemistry on porous silicon provides for ultrahigh sensitivity and analyte specificity with desorption/ionization on silicon mass spectrometry (DIOS-MS) analysis. Here, we report that the silylation of oxidized porous silicon offers a DIOS platform that is resistant to air oxidation and acid/base hydrolysis. Furthermore, surface modification with appropriate hydrophobic silanes allows analytes to absorb to the surface via hydrophobic interactions for direct analyte extraction from complex matrixes containing salts and other nonvolatile interferences present in the sample matrix. This enables rapid cleanup by simply spotting the sample onto the modified DIOS target and removing the liquid phase containing the interferences. This approach is demonstrated in the analysis of protein digests and metabolites in biofluids, as well as for the characterizing of inhibitors from their enzyme complex. An unprecedented detection limit of 480 molecules (800 ymol) for des-Arg(9)-bradykinin is reported on a pentafluorophenyl-functionalized DIOS chip.

Silylation chemistry on porous silicon provides for ultrahigh sensitivity and analyte specificity with desorption/ionization on silicon mass spectrometry (DIOS-MS) analysis. Here, we report that the silylation of oxidized porous silicon offers a DIOS platform that is resistant to air oxidation and acid/base hydrolysis. Furthermore, surface modification with appropriate hydrophobic silanes allows analytes to absorb to the surface via hydrophobic interactions for direct analyte extraction from complex matrixes containing salts and other nonvolatile interferences present in the sample matrix. This enables rapid cleanup by simply spotting the sample onto the modified DIOS target and removing the liquid phase containing the interferences. This approach is demonstrated in the analysis of protein digests and metabolites in biofluids, as well as for the characterizing of inhibitors from their enzyme complex. An unprecedented detection limit of 480 molecules (800 ymol) for des-Arg(9)-bradykinin is reported on a pentafluorophenyl-functionalized DIOS chip.

A tandem mass spectrometry--based approach is demonstrated for detecting a receptor for Ad37, one of the causative agents for epidemic keratoconjunctivitis. Partial purification of membrane glycoproteins was performed by using lectin-affinity chromatography and SDS-PAGE. Gel bands that were shown to bind Ad37 by using Viral Overlay Protein Blot Assay (VOPBA) were excised, proteolyzed and analyzed by using nanoLC-MS/MS to identify putative receptors contained in a mixture of proteins. Four candidate receptors were identified among approximately 50 proteins based on a search against a protein database. Inhibition of gene delivery mediated by an Ad37 vector, with antibodies against the glycoproteins identified by tandem mass spectrometry, strongly indicated that Membrane Cofactor Protein (MCP), a member of the complement regulatory family of proteins, is the receptor. This rapid and sensitive MS/MS-based strategy is perceived to have wide potential applications for the detection of viral receptors.

Subgroup D adenovirus (Ad) types 8, 19, and 37 (Ad8, -19, and -37, respectively) are causative agents of epidemic keratoconjunctivitis and genital tract infections. Previous studies showed that Ad37 binds to a 50-kDa membrane glycoprotein expressed on human ocular (conjunctival) cells. To identify and characterize the role of the 50-kDa glycoprotein in Ad37 infection, we partially purified this molecule from solubilized Chang C conjunctival cell membranes by using lentil lectin chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liquid chromatography coupled to nano-electrospray ionization-tandem mass spectrometry was subsequently used to identify four Ad37 receptor candidates: CD46, CD87, CD98, and CD147. Immunodepletion analyses demonstrated that the 50-kDa protein is identical to CD46 (also known as membrane cofactor protein). The Ad37, but not Ad5, fiber knob bound to the extracellular domain of CD46, demonstrating a direct interaction of an Ad37 capsid protein with CD46. An antibody specific for the N-terminal 19 amino acids of CD46 also blocked Ad37 infection of human cervical carcinoma and conjunctival cells, indicating a requirement for CD46 in infection. Finally, expression of a 50-kDa isoform of human CD46 in a CD46-null cell line increased cell binding by wild-type Ad37 and gene delivery by an Ad vector pseudotyped with the Ad37 fiber, but not by a vector bearing the Ad5 fiber. Together, these studies demonstrate that CD46 serves as an attachment receptor for Ad37 and shed further light on the cell entry pathway of subgroup D Ads.

Enzymes regulate biological processes through the conversion of specific substrates to products. Therefore, of fundamental interest for every enzyme is the elucidation of its natural substrates. Here, we describe a general strategy for identifying endogenous substrates of enzymes by untargeted liquid chromatography-mass spectrometry (LC-MS) analysis of tissue metabolomes from wild-type and enzyme-inactivated organisms. We use this method to discover several brain lipids regulated by the mammalian enzyme fatty acid amide hydrolase (FAAH) in vivo, including known signaling molecules (e.g., the endogenous cannabinoid anandamide) and a novel family of nervous system-enriched natural products, the taurine-conjugated fatty acids. Remarkably, the relative hydrolytic activity that FAAH exhibited for lipid metabolites in vitro was not predictive of the identity of specific FAAH substrates in vivo. Thus, global metabolite profiling establishes unanticipated connections between the proteome and metabolome that enable assignment of an enzyme's unique biochemical functions in vivo.

High-resolution 1H nuclear magnetic resonance (NMR) spectroscopy can be used to produce a biochemical fingerprint of low-molecular-weight metabolites from complex biological mixtures such as tissue extracts and biofluids. Changes in such fingerprint profiles can be used to characterize the effects of toxic insult in in vivo systems. The technique is nonselective and requires little sample preparation or derivatization. In the present study, earthworms (Eisenia veneta) were exposed to three different model xenobiotics by a standard filter paper contact test, and toxicant-induced biochemical changes were then investigated by characterizing the changes in endogenous metabolites visible in 600-MHz 1H NMR spectra of tissue extracts. The NMR spectral intensities were converted to discrete numerical values and tabulated in order to provide data matrices suitable for multivariate analysis. Principal component analysis showed that changes had occurred in the biochemical profiles relative to the undosed controls. The 2-fluoro-4-methylaniline-treated worms showed a decrease in a resonance from a compound identified as 2-hexyl-5-ethyl-3-furansulfonate using a combination of high-performance liquid chromatography (HPLC)-Fourier transform mass spectrometry (IonSpec, Lake Forest, CA, USA) and 1H and 13C NMR spectroscopy. An increase in inosine monophosphate was also observed. The 4-fluoroaniline-treated worms showed a decrease in maltose concentrations, and 3,5-difluoroaniline exerted the same effect as 2-fluoro-4-methylaniline but to a lesser extent. These changes could potentially be used as novel biomarkers of xenobiotic toxicity and could be used to determine the mechanism of action of other toxic chemicals.

High-resolution 1H nuclear magnetic resonance (NMR) spectroscopy can be used to produce a biochemical fingerprint of low-molecular-weight metabolites from complex biological mixtures such as tissue extracts and biofluids. Changes in such fingerprint profiles can be used to characterize the effects of toxic insult in in vivo systems. The technique is nonselective and requires little sample preparation or derivatization. In the present study, earthworms (Eisenia veneta) were exposed to three different model xenobiotics by a standard filter paper contact test, and toxicant-induced biochemical changes were then investigated by characterizing the changes in endogenous metabolites visible in 600-MHz 1H NMR spectra of tissue extracts. The NMR spectral intensities were converted to discrete numerical values and tabulated in order to provide data matrices suitable for multivariate analysis. Principal component analysis showed that changes had occurred in the biochemical profiles relative to the undosed controls. The 2-fluoro-4-methylaniline-treated worms showed a decrease in a resonance from a compound identified as 2-hexyl-5-ethyl-3-furansulfonate using a combination of high-performance liquid chromatography (HPLC)-Fourier transform mass spectrometry (IonSpec, Lake Forest, CA, USA) and 1H and 13C NMR spectroscopy. An increase in inosine monophosphate was also observed. The 4-fluoroaniline-treated worms showed a decrease in maltose concentrations, and 3,5-difluoroaniline exerted the same effect as 2-fluoro-4-methylaniline but to a lesser extent. These changes could potentially be used as novel biomarkers of xenobiotic toxicity and could be used to determine the mechanism of action of other toxic chemicals.