Endogenous metabolites have gained increasing interest over the past 5 years largely for their implications in diagnostic and pharmaceutical biomarker discovery. METLIN (http://metlin.scripps.edu), a freely accessible web-based data repository, has been developed to assist in a broad array of metabolite research and to facilitate metabolite identification through mass analysis. METLINincludes an annotated list of known metabolite structural information that is easily cross-correlated with its catalogue of high-resolution Fourier transform mass spectrometry (FTMS) spectra, tandem mass spectrometry (MS/MS) spectra, and LC/MS data.

METLIN: a metabolite mass spectral database.

Free Radical Lipid Peroxidation: Mechanisms and Analysis

Electrospray ionization mass spectrometry has been used to study protein interactions driven by noncovalent forces The gentleness of the electrospray ionization process allows intact protein complexes to be directly detected by mass spectrometry Evidence from the growing body of literature suggests that the ESI-MS observations for these weakly bound systems reflect, to some extent, the nature of the interaction found in the condensed phase Stoichiometry of the complex can be easily obtained from the resulting mass spectrum because the molecular weight of the complex is directly measured For the study of protein interactions, ESI-MS is complementary to other biophysical methods, such as NMR and analytical ultracentrifugation However, mass spectrometry offers advantages in speed and sensitivity The experimental variables that play a role in the outcome of ESI-MS studies of noncovalently bound complexes are reviewed Several applications of ESI-MS are discussed, including protein interactions with metal ions and nucleic acids and subunit protein structures (quaternary structure)

Studying noncovalent protein complexes by electrospray ionization mass spectrometry

Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.

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Baculovirus as versatile vectors for protein expression in insect and mammalian cells

Glycosylation is a common post-translational modification to cell surface and extracellular matrix (ECM) proteins as well as to lipids. As a result, cells carry a dense coat of carbohydrates on their surfaces that mediates a wide variety of cell-cell and cell-matrix interactions that are crucial to development and function. Because of the historical difficulties with the analysis of complex carbohydrate structures, a detailed understanding of their roles in biology has been slow to develop. Just as mass spectrometry has proven to be the core technology behind proteomics, it stands to play a similar role in the study of functional implications of carbohydrate expression, known as glycomics. This review summarizes the state of knowledge for the mass spectrometric analysis of oligosaccharides with regard to neutral, sialylated, and sulfated compound classes. Mass spectrometric techniques for the ionization and fragmentation of oligosaccharides are discussed so as to give the reader the background to make informed decisions to solve structure-activity relations in glycomics.

Mass spectrometry of oligosaccharides.

The extensive metabolism and administration of low doses of ethinylestradiol (EE) in preclinical animal species necessitates a sensitive analytical method to quantify the drug at low picogram-per-milliliter concentrations in biological matrixes. A highly sensitive and accurate method based on the derivatization of EE with dansyl chloride coupled with liquid chromatography/tandem mass spectrometry is described. The dansyl derivatization of EE introduced a basic secondary nitrogen into the molecule that was readily ionized in commonly used acidic HPLC mobile phases. The derivative showed an intense protonated molecular ion at m/z 530 under positive turbo ion spray ionization. The collision-induced dissociation of this ion formed a distinctive product at m/z 171, corresponding to the protonated 5-(dimethylamino)naphthalene moiety. The selected reaction monitoring, based on the m/z 530 → 171 transition, was highly specific for EE, since no background signal was observed from blank plasma obtained from rhesus ...

Derivatization of ethinylestradiol with dansyl chloride to enhance electrospray ionization: application in trace analysis of ethinylestradiol in rhesus monkey plasma.

A new type of quadrupole linear ion trap mass spectrometer, Q TRAP™ LC/MS/MS system (Q TRAP™), was evaluated for its performance in two studies: firstly, the in vitro metabolism of gemfibrozil in human liver microsomes, and, secondly, the quantification of propranolol in rat plasma. With the built-in information-dependent-acquisition (IDA) software, the instrument utilizes full scan MS in the ion trap mode and/or constant neutral loss scans as survey scans to trigger product ion scan (MS2) and MS3 experiments to obtain structural information of drug metabolites ‘on-the-fly’. Using this approach, five metabolites of gemfibrozil were detected in a single injection. This instrument combines some of the unique features of a triple quadrupole mass spectrometer, such as constant neutral loss scan, precursor ion scan and multiple reaction monitoring (MRM), together with the capability of a three-dimensional ion trap. Therefore, it becomes a powerful instrument for metabolite identification. The fast duty cycle in the ion trap mode allows the use of full product ion scan for quantification. For the quantification of propranolol, both MRM mode and full product ion scan in the ion trap mode were employed. Similar sensitivity, reproducibility and linearity values were established using these two approaches. The use of the product ion scan mode for quantification provided a convenient tool in selecting transitions for improving selectivity during the method development stage. Copyright © 2003 John Wiley & Sons, Ltd.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/rcm.1037

Use of a quadrupole linear ion trap mass spectrometer in metabolite identification and bioanalysis

Despite recent advances in the application of data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) to the identification of drug metabolites in complex biological matrixes, a prior knowledge of the likely routes of biotransformation of the therapeutic agent of interest greatly facilitates the detection and structural characterization of its metabolites. Thus, prediction of the [M + H]+ m/z values of expected metabolites allows for the construction of user-defined MS(n) protocols that frequently reveal the presence of minor drug metabolites, even in the presence of a vast excess of coeluting endogenous constituents. However, this approach suffers from inherent user bias, as a result of which additional \"survey scans\" (e.g., precursor ion and constant neutral loss scans) are required to ensure detection of as many drug-related components in the sample as possible. In the present study, a novel approach to this problem has been evaluated, in which knowledge-based predictions of metabolic pathways are first derived from a commercial database, the output from which is used to formulate a list-dependent LC/MS(n) data acquisition protocol. Using indinavir as a model drug, a substructure similarity search on the MDL metabolism database with a similarity index of 60% yielded 188 \"hits\", pointing to the possible operation of two hydrolytic, two N-dealkylation, three N-glucuronidation, one N-methylation, and several aromatic and aliphatic oxidation pathways. Integration of this information with data-dependent LC/MS(n) analysis using an ion trap mass spectrometer led to the identification of 18 metabolites of indinavir following incubation of the drug with human hepatic postmitochondrial preparations. This result was accomplished with only a single LC/MS(n) run, representing significant savings in instrument use and operator time, and afforded an accurate view of the complex in vitro metabolic profile of this drug.

Integration of Knowledge-Based Metabolic Predictions with Liquid Chromatography Data-Dependent Tandem Mass

Tracking problems and possible solutions in the quantitative determination of small molecule drugs and metabolites in biological fluids using liquid chromatography-mass spectrometry

https://link.springer.com/content/pdf/10.1016/S1044-0305(03)00201-0.pdf

Ray Bakhtiar

Papers

Derivatization of ethinylestradiol with dansyl chloride to enhance electrospray ionization: application in trace analysis of ethinylestradiol in rhesus monkey plasma.

Use of a quadrupole linear ion trap mass spectrometer in metabolite identification and bioanalysis

Integration of Knowledge-Based Metabolic Predictions with Liquid Chromatography Data-Dependent Tandem Mass

Tracking problems and possible solutions in the quantitative determination of small molecule drugs and metabolites in biological fluids using liquid chromatography-mass spectrometry

Detection and characterization of methionine oxidation in peptides by collision-induced dissociation and electron capture dissociation.