Ion mobility–mass spectrometry for structural proteomics

Ion mobility coupled to mass spectrometry has been an important tool in the fields of chemical physics and analytical chemistry for decades, but its potential for interrogating the structure of proteins and multiprotein complexes has only recently begun to be realized. Today, ion mobility–mass spectrometry is often applied to the structural elucidation of protein assemblies that have failed high-throughput crystallization or NMR spectroscopy screens. Here, we highlight the technology, approaches and data that have led to this dramatic shift in use, including emerging trends such as the integration of ion mobility–mass spectrometry data with more classical (e.g., ‘bottom-up’) proteomics approaches for the rapid structural characterization of protein networks.     

Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

Gas chromatography — mass spectrometry of catechol estrogens

The gas chromatographic and mass spectrometric properties of 19 catechol estrogens and catechol estrogen methyl ethers are reported. The gas chromatographic behaviour of the TMS-derivatives on the stationary phases OV-1, OV-3, OV-7, and OV-17 is examined and correlated with their molecular weight, shape, and polarity. The characteristic mass spectrometric features of the compounds result from the aromatic ring A, which is able to stabilize positive charge within the molecular ions. Consequently the molecular ions form the base peaks of the spectra. Fragmentation patterns highly specific for the catechols as well as for their monomethyl and dimethyl ethers are discussed and substantiated by determination of metastable ions and high resolution mass measurements.     

Dissecting intercellular signaling with mass spectrometry–based proteomics

Physiological functions depend on a coordinated interplay of numerous different cell types. Proteins serve as major signaling molecules between cells; however, their comprehensive investigation in physiologically relevant settings has remained challenging. Mass spectrometry (MS)–based shotgun proteomics is emerging as a powerful technology for the systematic analysis of protein-mediated intercellular signaling and regulated post-translational modifications. Here, we discuss recent advancements in cell biological, chemical, and biochemical MS-based approaches for the profiling of cellular messengers released by sending cells, receptors expressed on the cell surface, and their interactions. We highlight methods tailored toward the mapping of dynamic signal transduction mechanisms at cellular interfaces and approaches to dissect communication cell specifically in heterocellular systems. Thereby, MS-based proteomics contributes a unique systems biology perspective for the identification of intercellular signaling pathways deregulated in disease.     

The mass—Energy balance of anaerobic methane production

A theory was developed for the mass and energy balance of microbial processes, with special reference to the anaerobic production of methane. Interrelations of the bioengineering parameters of the process were delineated substrate quality, biodegrability and biological effciency of anaerobic processing of complex organic waste substrates. Application of the method is demonstrated on practical examples.     

Next-generation capillary electrophoresis–mass spectrometry approaches in metabolomics

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Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

Fast liquid chromatographic–mass spectrometric determination of pharmaceutical compounds

We present fast LC–MS–MS analyses of multicomponent mixtures containing flavones, sulfonamides, benzodiazepines and tricyclic amines. Using a short microbore HPLC column with small particle size, five to eight compounds were partially resolved within 15 to 30 s. TurboIonSpray and atmospheric pressure chemical ionization interfaces were well suited to tolerate the higher eluent flow-rates of 1.2 to 2 ml/min. The methods were applied to biological sample matrices after clean-up using solid-phase or liquid–liquid extraction. Good precision and accuracy (average 8.9 and 97.7%, respectively) were achieved for the determination of tricyclic amines in human plasma. Benzodiazepines were determined in human urine with average precision of 9% and average accuracy of 95% for intra- and inter-assay. Detection limits in the low ng/ml range were obtained. An example for 240 injections per hour of demonstrated the feasibility of rapid LC–MS–MS analysis.     

Ethnocentricity and the social construction of ‘mass hysteria’

This study provides a critical historical review and analysis of the variety of human expressions which have been erroneously labeled under the grandiose category mass hysteria. It is argued that Western science reductionist approaches to the classification of mass hysteria treat it as an entity to be discovered transculturally, and in their self-fulfilling search for universals systematically exclude what does not fit within the autonomous parameters of its Western-biased culture model, exemplifying what Kleinman (1977) terms a category fallacy. As a result of objectivist methodologies, the etiology of actions labeled as mass hysteria is typically viewed as deviant, irrational or abnormal behavior resulting from a malfunctioning proper social order. However, what constitutes the correct social order is a function of a researcher's historical sociocultural and/or scientific milieu. This study reviews the problem, advocating Geertz's (1973) culturally relativistic approach to understanding various cross-cultural behavior that is sensitive to and tolerant of the unique context and milieu of participants. Mass or epidemic hysteria is viewed as an invention of Western psychiatry and should be abandoned and replaced with the term collective exaggerated emotions. Instead of attempting to discover a neatly packaged, unitary external disease entity, the focus of a meaning-oriented approach emphasizes the deciphering of foreign realities, semantic networks and symbol systems.     

A mass spectrometry-based method to screen for α-amidated peptides

Amidation is a post-translational modification found at the C-terminus of ~50% of all neuropeptide hormones. Cleavage of the C(α)-N bond of a C-terminal glycine yields the α-amidated peptide in a reaction catalyzed by peptidylglycine α-amidating monooxygenase (PAM). The mass of an α-amidated peptide decreases by 58 Da relative to its precursor. The amino acid sequences of an α-amidated peptide and its precursor differ only by the C-terminal glycine meaning that the peptides exhibit similar RP-HPLC properties and tandem mass spectral (MS/MS) fragmentation patterns. Growth of cultured cells in the presence of a PAM inhibitor ensured the coexistence of α-amidated peptides and their precursors. A strategy was developed for precursor and α-amidated peptide pairing (PAPP): LC-MS/MS data of peptide extracts were scanned for peptide pairs that differed by 58 Da in mass, but had similar RP-HPLC retention times. The resulting peptide pairs were validated by checking for similar fragmentation patterns in their MS/MS data prior to identification by database searching or manual interpretation. This approach significantly reduced the number of spectra requiring interpretation, decreasing the computing time required for database searching and enabling manual interpretation of unidentified spectra. Reported here are the α-amidated peptides identified from AtT-20 cells using the PAPP method.     

Liquid chromatography–mass spectrometry in forensic and clinical toxicology

This paper reviews liquid chromatographic–mass spectrometric (LC–MS) procedures for the identification and/or quantification of drugs of abuse, therapeutic drugs, poisons and/or their metabolites in biosamples (whole blood, plasma, serum, urine, cerebrospinal fluid, vitreous humor, liver or hair) of humans or animals (cattle, dog, horse, mouse, pig or rat). Papers published from 1995 to early 1997, which are relevant to clinical toxicology, forensic toxicology, doping control or drug metabolism and pharmacokinetics, were taken into consideration. They cover the following analytes: amphetamines, cocaine, lysergide (LSD), opiates, anabolics, antihypertensives, benzodiazepines, cardiac glycosides, corticosteroids, immunosuppressants, neuroleptics, non-steroidal anti-inflammatory drugs (NSAID), opioids, quaternary amines, xanthins, biogenic poisons such as aconitines, aflatoxins, amanitins and nicotine, and pesticides. LC–MS interface types, mass spectral detection modes, sample preparation procedures and chromatographic systems applied in the reviewed papers are discussed. Basic information about the biosample assayed, work-up, LC column, mobile phase, interface type, mass spectral detection mode, and validation data of each procedure is summarized in tables. Examples of typical LC–MS applications are presented.     

Analysing body condition: mass, volume or density?

1. Body condition (defined as the relative amount of energy reserves in the body) is an animal trait with strong ecological implications. In some animal taxa (e.g. arthropods), the external volume of the body part in which most nutrients are stored (e.g. abdomen) is used interchangeably with body mass to estimate body condition, making the implicit assumption that abdomen residual volume is a good surrogate of residual mass. However, the degree of correlation between these two measures should largely depend on the density of the nutrients stored. 2. We simulated two food-supplemented experimental groups of animals, each storing a slightly different amount of lipids either in their abdomens or in their entire bodies, and explored (i) how different estimates of condition were able to detect fixed differences between the groups; and (ii) how the amount of lipids stored could affect the outcome of non-intrusive measures of condition on a dichotomous variable (e.g. survival, mating success). We found that density body condition (body mass statistically controlled for structural body size and body volume) has much greater power to detect differences between experimental groups or effects on binary response variables than do classic mass/size or volume/size condition indices. 3. Using data on Lycosa tarantula (L.), a burrowing wolf spider, we report dramatic differences among these three indices in their ability to detect sex differences in the effect of feeding treatment on body condition at maturity. In particular, a plot of residual mass against residual volume reflecting nutrient density suggests that poorly fed spiders are nutritionally unbalanced, since well-fed spiders invest in nutrients of very different density. 4. Furthermore, using data on Scathophaga stercoraria (L.), the yellow dung fly, we found that an index of density condition was better at distinguishing condition differences among three populations than were mass or volume condition estimates alone. 5. We propose that including these three surrogates of condition (mass, volume and density) will substantially improve the accuracy of non-intrusive estimates of body condition, thus providing more powerful tools with direct application in a wide range of disciplines.     

Detection of estradiol-17β during a mass coral spawn

The steroid estradiol-17 (E₂) is associated with female gametogenesis in all vertebrates and many invertebrates. This is the first report of estrogens in scleractinian corals. Seawater and egg slicks were collected during a mass coral spawn at Ningaloo reef, Western Australia for the measurement of total phosphate (TP) and E₂. Total P in the water column increased 600 times, from 0.5M to 300M. Concentrations of E₂ increased nearly 8 fold during the spawn, from 55 to 420 pg/100 ml seawater. Coral eggs collected from egg slicks contained 368±40 pg E₂/g dry wt of eggs. Estrogen may be a key hormone in a simple endocrine system of scleractinian corals that synchronizes growth and development of coral oocytes. Its potential role in triggering spawning via chemical messengers in the water column warrants further research.     

mTORC1 signaling and regulation of pancreatic β-cell mass

The capacity of β cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of β cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for β-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of β-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in β cells. This review will discuss recent advances in the understanding of how this pathway regulates β-cell mass and present data on the role of TSC1 in modulation of β-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic β cells results in improved glucose tolerance, hyperinsulinemia and expansion of β-cell mass that persists with aging.     

Is mass spectrometry ready for proteome-wide protein expression analysis?

Recent advances in mass spectrometry will soon allow routine analysis of protein expression levels. How close are we to true quantitative proteomics?     

mTORC1 signaling and regulation of pancreatic β-cell mass

The capacity of β cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of β cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for β-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of β-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in β cells. This review will discuss recent advances in the understanding of how this pathway regulates β-cell mass and present data on the role of TSC1 in modulation of β-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic β cells results in improved glucose tolerance, hyperinsulinemia and expansion of β-cell mass that persists with aging.     

Differentiation of α-hemolytic Streptococci by direct mass spectrometry profiling

The modern phenotypic and genetic methods except for Multi Locus Sequence Typing do not allow the reliable differentiation within Mitis group of α-hemolytic streptococci. During this study the MALDI mass spectra were acquired for 28 clinical isolates initially identified as S. pneumoniae by routine bacteriological tests. Due to Multi Locus Sequence Typing these isolates were found to belong to two closely related species - S. pneumoniae (n = 22) and S. mitis (n = 6). Distribution of those isolates in accordance with cluster analysis of collected mass spectra matched to Multi Locus Sequence Typing data. The diagnostic model based on Genetic Algorithm classifier demonstrated the differentiation of α-hemolytic streptococci with 100% sensitivity and 94.6% accuracy. Statistical analysis of MS peak areas revealed 2 peaks which are different for S. mitis and S. pneumoniae groups.