Chemical characterization of a family of brain lipids that induce sleep.

A molecule isolated from the cerebrospinal fluid of sleep-deprived cats has been chemically characterized and identified as cis-9,10-octadecenoamide. Other fatty acid primary amides in addition to cis-9,10-octadecenoamide were identified as natural constituents of the cerebrospinal fluid of cat, rat, and human, indicating that these compounds compose a distinct family of brain lipids. Synthetic cis-9,10-octadecenoamide induced physiological sleep when injected into rats. Together, these results suggest that fatty acid primary amides may represent a previously unrecognized class of biological signaling molecules.

XCMS2: processing tandem mass spectrometry data for metabolite identification and structural characterization.

Mass spectrometry based metabolomics represents a new area for bioinformatics technology development. While the computational tools currently available such as XCMS statistically assess and rank LC-MS features, they do not provide information about their structural identity. XCMS(2) is an open source software package which has been developed to automatically search tandem mass spectrometry (MS/MS) data against high quality experimental MS/MS data from known metabolites contained in a reference library (METLIN). Scoring of hits is based on a "shared peak count" method that identifies masses of fragment ions shared between the analytical and reference MS/MS spectra. Another functional component of XCMS(2) is the capability of providing structural information for unknown metabolites, which are not in the METLIN database. This "similarity search" algorithm has been developed to detect possible structural motifs in the unknown metabolite which may produce characteristic fragment ions and neutral losses to related reference compounds contained in METLIN, even if the precursor masses are not the same.

Biochemical mass spectrometry: worth the weight?

The utility of mass spectrometry for the analysis of biological molecules has been enhanced by the development of two techniques that generate gas-phase ions via nondestructive vaporization and ionization. These techniques can be used not only to determine the primary structure of biological molecules with unprecedented accuracy, but also to map noncovalent biomolecular interactions.

Quantitative electrospray mass spectrometry for the rapid assay of enzyme inhibitors.

BACKGROUND: Combinatorial chemistry has become an important method for identifying effective ligand-receptor binding, new catalysts and enzyme inhibitors. In order to distinguish the most active component of a library or to obtain structure-activity relationships of compounds in a library, an efficient quantitative assay is crucial. Electrospray mass spectrometry has become an indispensable tool for qualitatively screening combinatorial libraries and its use for quantitative analysis has recently been demonstrated. RESULTS: This paper describes the use of quantitative electrospray mass spectrometry for screening libraries of inhibitors of enzymatic reactions, specifically the enzymatic glycosylation by beta-1,4-galactosyltransferase, which catalyzes the transfer of galactose from uridine-5'-diphosphogalactose to the 4-position of N-acetylglucosamine beta OBn (Bn: benzene) to form N-acetyllactosamine beta OBn. Our mass spectrometric screening approach showed that both nucleoside diphosphates and triphosphates inhibited galactosyltransferase while none of the nucleoside monophosphates, including uridine-5'-monophosphate, showed any inhibition. Additional libraries were generated in which the concentrations of the inhibitors were varied and, using mass spectrometry, uridine-5'-diphosphate-2-deoxy-2-fluorogalactose was identified as the best inhibitor. CONCLUSIONS: This report introduces quantitative electrospray mass spectrometry as a rapid, sensitive and accurate quantitative assaying tool for inhibitor libraries that does not require a chromophore or radiolabeling. A viable alternative to existing analytical techniques is thus provided. The new technique will greatly facilitate the discovery of novel inhibitors against galactosyltransferase, an enzyme for which there are few potent inhibitors.

Mass spectrometry and viral analysis.

BACKGROUND: Electrospray ionization (ESI) mass spectrometry is a powerful new approach for analyzing biomolecules and biomolecular complexes. Previous studies have provided evidence that non-covalent biomolecular complexes can be observed by ESI mass spectrometry; it is not clear, however, whether the native conformation of the biomolecules is maintained throughout the ionization and analysis process. We set out to address this question using live viruses. RESULTS: Viral ions have been generated in the gas phase using electrospray ionization mass spectrometry. These ions have been collected, following ion filtering through the mass analyzer, and then analyzed by transmission electron microscopy. Transmission electron microscopy revealed that rice yellow mottle virus and tobacco mosaic virus retained their respective spherical and rod-like ultrastructure. The viability of the isolated tobacco mosaic virus was confirmed by inoculation and infection of tobacco plants. CONCLUSIONS: These results demonstrate the utility of electrospray for supramolecular complexes with molecular weights of over 40 million Da and offer conclusive evidence that native biomolecular structures can be conserved through the electrospray process.

Quench-flow experiments combined with mass spectrometry show apomyoglobin folds through and obligatory intermediate.

Folding of apomyoglobin is characterized by formation of a compact intermediate that contains substantial helicity. To determine whether this intermediate is obligatory or whether the protein can fold directly into the native state via an alternate parallel pathway, we have combined quench-flow hydrogen-exchange pulse labeling techniques with electrospray ionization mass spectrometry. The mass spectra of apomyoglobin obtained at various refolding times suggest that apomyoglobin indeed folds through a single pathway containing an obligatory intermediate with a significant hydrogen-bonded secondary structure content.

Probing viruses with mass spectrometry.

Mass spectrometry offers a new perspective on the solution and gas-phase properties of viruses. Its broad application to local and global viral structure provides unique insights into many biological processes, including viral-antibody binding, protein-protein interactions and protein dynamics. Mass measuring viral proteins is now routine and since viruses are typically well characterized, in that the capsid proteins and DNA (or RNA) sequences are known, identifying a virus based on the mass of the protein and enzymatic digestion fragments is relatively straightforward. Using mass spectrometry, this paper describes the identification of viral protein post-translational modifications such as myristoylation, phosphorylation and disulfide bridging. Furthermore, complementary data obtained with mass spectrometry and x-ray crystallography demonstrate that viruses are highly dynamic particles whose viral capsid's mobility could, until recently, be inferred only from inherently static spectroscopic methods. Lastly, mass spectrometry has been applied on a global scale via the mass measurement of entire intact viruses. Given the general utility of mass spectrometry, its continuing development should further its application to viral dynamics, structure, function and identification.

Probing protein structure using biochemical and biophysical methods. Proteolysis, matrix-assisted laser desorption/ionization mass spectrometry, high-performance liquid chromatography and size-exclusion chromatography of p21Waf1/Cip1/Sdi1.

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53 tumor suppressor-dependent cell growth control in humans and other organisms, mediates G1/S-phase arrest through inhibition of cyclin-dependent kinases (Cdks). The enzymatic activity of these kinases is essential for progress through the cell division cycle and one level of cell cycle regulation is exerted through inhibition of Cdks by a family of small proteins, including p21. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2-terminus. Using proteolytic mapping, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, HPLC and size-exclusion chromatography, we show that p21, active as a Cdk inhibitor, exists in an extended, non-globular conformation in the absence of its biological target and that p21 lacks the hallmarks of stable secondary and tertiary structure. We have developed an efficient approach to obtain detailed proteolytic maps that takes advantage of the high accuracy and sensitivity of MALDI mass spectrometry. Our method allows a proteolytic map to be obtained from a single mass spectrum for fragments produced from a single proteolytic reaction.

Electrospray and MALDI mass spectrometry in the identification of spermicides in criminal investigations.

Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have been used to examine evidence in a sexual assault investigation. Because condoms are being used increasingly by sexual assailants and some condom brands include the spermicide nonoxynol-9 (nonylphenoxy polyethoxyethanol) in the lubricant formulation, the recovery, and identification of nonoxynol-9 from evidence items may assist in proving corpus delicti. A method was developed for the recovery of nonoxynol-9 from internal vaginal swabs and for its identification by reverse phase liquid chromatography/electrospray ionization mass spectrometry (LC ESI-MS), nanoelectrospray ionization (nanoESI) mass spectrometry, and high resolution MALDI Fourier transform mass spectrometry (MALDI-FTMS). The method was tested on extracts from precoitus, immediate postcoitus, and four-hours postcoitus vaginal swabs provided by a volunteer whose partner does not normally use condoms, but for this trial used a condom having a water-soluble gel-type lubricant that includes 5% nonoxynol-9 in its formulation. Subsequently, LC ESI-MS was used to identify traces of nonoxynol-9 from the internal vaginal swab of a victim of a sexual assault.

Nanostructure-initiator mass spectrometry (NIMS) imaging of brain cholesterol metabolites in Smith-Lemli-Opitz syndrome.

Cholesterol is an essential component of cellular membranes that is required for normal lipid organization and cell signaling. While the mechanisms associated with maintaining cholesterol homeostasis in the plasma and peripheral tissues have been well studied, the role and regulation of cholesterol biosynthesis in normal brain function and development have proven much more challenging to investigate. Smith-Lemli-Opitz syndrome (SLOS) is a disorder of cholesterol synthesis characterized by mutations of 7-dehydrocholesterol reductase (DHCR7) that impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol and lead to neurocognitive deficits, including cerebellar hypoplasia and austism behaviors. Here we have used a novel mass spectrometry-based imaging technique called cation-enhanced nanostructure-initiator mass spectrometry (NIMS) for the in situ detection of intact cholesterol molecules from biological tissues. We provide the first images of brain sterol localization in a mouse model for SLOS (Dhcr7(-/-)). In SLOS mice, there is a striking localization of both 7DHC and residual cholesterol in the abnormally developing cerebellum and brainstem. In contrast, the distribution of cholesterol in 1-day old healthy pups was diffuse throughout the cerebrum and comparable to that of adult mice. This study represents the first application of NIMS to localize perturbations in metabolism within pathological tissues and demonstrates that abnormal cholesterol biosynthesis may be particularly important for the development of these brain regions.

Desorption/ionization on silicon nanowires.

Dense arrays of single-crystal silicon nanowires (SiNWs) have been used as a platform for laser desorption/ionization mass spectrometry of small molecules, peptides, protein digests, and endogenous and xenobiotic metabolites in biofluids. Sensitivity down to the attomole level has been achieved on the nanowire surfaces by optimizing laser energy, surface chemistry, nanowire diameter, length, and growth orientation. An interesting feature of the nanowire surface is that it requires lower laser energy as compared to porous silicon and MALDI to desorb/ionize small molecules, therefore reducing background ion interference. Taking advantage of their high surface area and fluid wicking capabilities, SiNWs were used to perform chromatographic separation followed by mass analysis of the separated molecules providing a unique platform that can integrate separation and mass spectrometric detection on a single surface.

The emergence of mass spectrometry in biochemical research.

The initial steps toward routinely applying mass spectrometry in the biochemical laboratory have been achieved. In the past, mass spectrometry was confined to the realm of small, relatively stable molecules; large or thermally labile molecules did not survive the desorption and ionization processes intact. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allow for the analysis of both small and large biomolecules through "mild" desorption and ionization methods. The use of ESI and MALDI mass spectrometry extends beyond simple characterization. Noncovalent interactions, protein and peptide sequencing, DNA sequencing, protein folding, in vitro drug analysis, and drug discovery are among the areas to which ESI and MALDI mass spectrometry have been applied. This review summarizes recent developments and major contributions in mass spectrometry, focusing on the applications of MALDI and ESI mass spectrometry.

Cationic drug analysis using matrix-assisted laser desorption/ionization mass spectrometry: application to influx kinetics, multidrug resistance, and intracellular chemical change.

Highly sensitive and convenient analysis of intracellular cationic drugs has been achieved by applying matrix-assisted laser desorption/ionization mass spectrometry (MALD-MS). Tetraphenylphosphonium cation was readily identified and quantified (using methyltriphenylphosphonium cation as an internal standard) at subpicomole levels in crude lysate from < 4 x 10(3) FaDu human hypopharyngeal carcinoma cells. A quantitative MALD-MS time course for tetraphenylphosphonium cation accumulation into FaDu cells was comparable to a time course using scintillation counting with tritiated tetraphenylphosphonium. MALD-MS was also capable of demonstrating the reduced accumulation of the cationic drug rhodamine-123 by DoxR MCF7, a multiply drug-resistant human breast adenocarcinoma cell line, relative to the nonresistant parent line MCF7. In addition, MALD-MS was used to follow a chemical reaction inside intact FaDu cells: the formation of a hydrazone (II-51) from benzaldehyde and an acylhydrazide, 5-[tris(4-dimethylaminophenyl)phosphonio]pentanoyl hydrazide (II-25). These results suggest that MALD-MS may provide a rapid and practical alternative to existing methods for the analysis of cationic drugs, toxins, and their metabolites in cells and tissues.

Applications of mass spectrometry in combinatorial chemistry.

This article describes the use of two mass spectrometric techniques, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry, toward a variety of challenging problems in drug discovery and identification. Quantitative ESI was used to screen for inhibitor activity of two different enzymatic glycosylation reactions resulting in the identification of the most effective inhibitors and the determination of their IC50 (inhibitor concentration at 50% inhibition). Also described is a combinatorial extraction method used with automated MALDI mass spectrometry to improve upon the clinical analysis of the immunosuppressant drug cyclosporin A (CsA). Optimization was performed by generating an array of solvent systems which were screened (by MALDI-MS) for the most efficient extraction of CsA from whole blood. Ultimately a 70/30 hexane:CHCl3 mixture was identified as the most efficient binary solvent system for such extractions. In addition it was demonstrated that peptides and carbohydrates, covalently linked to a polymeric support (through a photolabile linker), can be directly analyzed by MALDI in a single step which requires no pretreatment of the sample to induce cleavage from the support. The UV laser light in the MALDI experiment was used to simultaneously promote the analyte's photolytic cleavage from the solid support and its gas phase ionization for subsequent mass spectral analysis. Overall, the strength of mass spectrometry lies in its versatility, making it a powerful analytical technique with which to characterize the diversity of compounds found in combinatorial libraries.

An automated MALDI mass spectrometry approach for optimizing cyclosporin extraction and quantitation.

A combinatorial extraction method and an automated matrix-assisted laser desorption/ionization (MALDI) mass spectrometry procedure were used to improve the clinical analysis of the immunosuppressant drug cyclosporin A. Cyclosporin extracts from whole blood were analyzed by MALDI and electrospray ionization (ESI) mass spectrometry, allowing for their identification and quantification. Due to limitations associated with the current multistep cyclosporin extraction procedure from whole blood, a combinatorial approach was devised to optimize this extraction. Optimization was performed by generating an array of solvent systems to be used for extraction from blood, and an automated analysis was carried out on a MALDI mass spectrometer to identify successful extractions. The first generation of experiments revealed four binary solvent systems to be effective for cyclosporin extraction (hexane/EtOH, ACN/H2O, ACN/MeOH, and hexane/CHCl3). A new array based on these solvent systems was generated, and a second iteration of these experiments was then performed. In the second generation of experiments, hexane/CHCl3 (70:30) was found to provide the most effective single-step extraction of these solvent systems for cyclosporin and its metabolites. The limits of detection were determined to be 15 ng/mL in whole blood for ESI/MS and MALDI-MS and could also be used for identifying major drug metabolites. In addition to applying this combinatorial approach to extraction procedures, this experimental design could easily be extended to examine other approaches, such as optimizing chemical reactions and screening inhibitors in enzymatic reactions.

Mass spectrometry in viral proteomics.

Mass spectrometry is a valuable tool in structural and functional viral proteomics, where it has been used to identify viral capsid proteins, viral mutants, and posttranslational modifications. Further, mass-based approaches combined with time-resolved proteolysis (mass mapping) have revealed the dynamic nature of viral particles in solution; this method is contributing to an understanding of the dynamic domains of the viral capsid which may have significant value in developing new approaches for viral inactivation. As a result of these experiments, and by comparison with complementary data from X-ray crystallography, a new dimension to viral protein structure and function is emerging.

Desorption-ionization mass spectrometry on porous silicon.

Desorption mass spectrometry has undergone significant improvements since the original experiments were performed more than 90 years ago. The most dramatic change occurred in the early 1980s with the introduction of an organic matrix to transfer energy to the analyte. This reduces ion fragmentation but also introduces background ions from the matrix. Here we describe a matrix-free strategy for biomolecular mass spectrometry based on pulsed-laser desorption-ionization from a porous silicon surface. Our method uses porous silicon to trap analytes deposited on the surface, and laser irradiation to vaporize and ionize them. We show that the method works at femtomole and attomole levels of analyte, and induces little or no fragmentation, in contrast to what is typically observed with other such approaches. The ability to perform these measurements without a matrix also makes it more amenable to small-molecule analysis. Chemical and structural modification of the porous silicon has enabled optimization of the ionization characteristics of the surface. Our technique offers good sensitivity as well as compatibility with silicon-based microfluidics and microchip technologies.

Desorption/ionization on silicon (DIOS): a diverse mass spectrometry platform for protein characterization.

Since the advent of matrix-assisted laser desorption/ionization and electrospray ionization, mass spectrometry has played an increasingly important role in protein functional characterization, identification, and structural analysis. Expanding this role, desorption/ionization on silicon (DIOS) is a new approach that allows for the analysis of proteins and related small molecules. Despite the absence of matrix, DIOS-MS yields little or no fragmentation and is relatively tolerant of moderate amounts of contaminants commonly found in biological samples. Here, functional assays were performed on an esterase, a glycosidase, a lipase, as well as exo- and endoproteases by using enzyme-specific substrates. Enzyme activity also was monitored in the presence of inhibitors, successfully demonstrating the ability of DIOS to be used as an inhibitor screen. Because DIOS is a matrix-free desorption technique, it also can be used as a platform for multiple analyses to be performed on the same protein. This unique advantage was demonstrated with acetylcholine esterase for qualitative and quantitative characterization and also by its subsequent identification directly from the DIOS platform.

Monitoring enzyme catalysis with mass spectrometry.

Mass spectrometry is a rapid, sensitive, and accurate quantitative approach for the direct monitoring of enzyme-catalyzed reactions that does not require a chromophore or radiolabeling and thus provides a viable alternative to existing analytical techniques. In this study the proteolysis of intact viral capsid proteins, the alpha-glucosidase-catalyzed hydrolysis of p-nitrophenyl-alpha-glucopyranoside and the lipoprotein lipase-catalyzed ester hydrolysis of resorufin were examined. Matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry were used to examine the proteolysis of viral protein capsids, providing information about capsid dynamics and the stabilizing force of viral protein/RNA interactions. In addition, k(cat) and K(m) values of enzyme-catalyzed hydrolysis were obtained (without the use of a chromophore). These results also demonstrate the effect an unnatural substrate can have on enzyme activity. Overall, mass spectrometry provides for efficient and quantitative analysis of enzyme-catalyzed reactions, as well as the direct observation of reaction dynamics.