Impact of Azithromycin on the Quorum Sensing-Controlled Proteome of Pseudomonas aeruginosa.

The macrolide antibiotic, azithromycin (AZM), has been reported to improve the clinical outcome of cystic fibrosis patients, many of whom are chronically-infected with Pseudomonas aeruginosa. However, the highest clinically-achievable concentrations of this drug are well-below the minimum inhibitory concentration for P. aeruginosa, raising the question of why AZM exhibits therapeutic activity. One possibility that has been raised by earlier studies is that AZM inhibits quorum sensing (QS) by P. aeruginosa. To explicitly test this hypothesis the changes brought about by AZM treatment need to be compared with those associated with specific QS mutants grown alongside in the same growth medium, but this has not been done. In this work, we used quantitative 2D-difference gel electrophoresis and 1H-NMR spectroscopy footprint analysis to examine whether a range of clinically-relevant AZM concentrations elicited proteomic and metabolomic changes in wild-type cultures that were similar to those seen in cultures of defined QS mutants. Consistent with earlier reports, over half of the AZM-induced spot changes on the 2D gels were found to affect QS-regulated proteins. However, AZM modulated very few protein spots overall (compared with QS) and collectively, these modulated proteins comprised only a small fraction (12-13%) of the global QS regulon. We conclude that AZM perturbs a sub-regulon of the QS system but does not block QS per se. Reinforcing this notion, we further show that AZM is capable of attenuating virulence factor production in another Gram-negative species that secretes copious quantities of exoenzymes (Serratia marcescens), even in the absence of a functional QS system.

Sex-Related Differences in Rat Choroid Plexus and Cerebrospinal Fluid: A cDNA Microarray and Proteomic Analysis.

The choroid plexus (CP) epithelium is a unique structure in the brain that forms an interface between the peripheral blood and the cerebrospinal fluid (CSF), which is mostly produced by the CP itself. Because the CP transcriptome is regulated by the sex hormone background, the present study compared gene/protein expression profiles in the CP and CSF from male and female rats aiming to better understand sex-related differences in CP functions and brain physiology. We used data previously obtained by cDNA microarrays to compare the CP transcriptome between male and female rats, and complemented these data with the proteomic analysis of the CSF of castrated and sham-operated males and females. Microarray analysis showed that 17 128 and 17 002 genes are expressed in the male and female CP, which allowed the functional annotation of 141 and 134 pathways, respectively. Among the most expressed genes, canonical pathways associated with mitochondrial dysfunctions and oxidative phosphorylation were the most prominent, whereas the most relevant molecular and cellular functions annotated were protein synthesis, cellular growth and proliferation, cell death and survival, molecular transport, and protein trafficking. No significant differences were found between males and females regarding these pathways. Seminal functions of the CP differentially regulated between sexes were circadian rhythm signalling, as well as several canonical pathways related to stem cell differentiation, metabolism and the barrier function of the CP. The proteomic analysis identified five down-regulated proteins in the CSF samples from male rats compared to females and seven proteins exhibiting marked variation in the CSF of gonadectomised males compared to sham animals, whereas no differences were found between sham and ovariectomised females. These data clearly show sex-related differences in CP gene expression and CSF protein composition that may impact upon neurological diseases.

Remodelling of a polypyrimidine tract-binding protein complex during apoptosis activates cellular IRESs.

Post-transcriptional control of gene expression is mediated by the interaction of RNA-binding proteins with their cognate mRNAs that specifically regulate their stability, localization and translation. mRNA-binding proteins are multifunctional and it has been proposed therefore that a combinatorial RNA-binding protein code exists that allows specific protein sub-complexes to control cytoplasmic gene expression under a range of pathophysiological conditions. We show that polypyrimidine tract-binding protein (PTB) is central to one such complex that forms in apoptotic cells. Thus, during apoptosis initiated by TNF-related apoptosis inducing ligand there is a change in the repertoire of RNA-binding proteins with which PTB interacts. We show that altering the cellular levels of PTB and its binding partners, either singly or in combination, is sufficient to directly change the rates of apoptosis with increased expression of PTB, YBX1, PSF and NONO/p54(nrb) accelerating this process. Mechanistically, we show that these proteins post-transcriptionally regulate gene expression, and therefore apoptotic rates, by interacting with and stimulating the activity of RNA elements (internal ribosome entry segments) found in mRNAs that are translated during apoptosis. Taken together, our data show that PTB function is controlled by a set of co-recruited proteins and importantly provide further evidence that it is possible to dictate cell fate by modulating cytoplasmic gene expression pathways alone.

The effect of organelle discovery upon sub-cellular protein localisation.

Prediction of protein sub-cellular localisation by employing quantitative mass spectrometry experiments is an expanding field. Several methods have led to the assignment of proteins to specific subcellular localisations by partial separation of organelles across a fractionation scheme coupled with computational analysis. Methods developed to analyse organelle data have largely employed supervised machine learning algorithms to map unannotated abundance profiles to known protein-organelle associations. Such approaches are likely to make association errors if organelle-related groupings present in experimental output are not included in data used to create a protein-organelle classifier. Currently, there is no automated way to detect organelle-specific clusters within such datasets. In order to address the above issues we adapted a phenotype discovery algorithm, originally created to filter image-based output for RNAi screens, to identify putative subcellular groupings in organelle proteomics experiments. We were able to mine datasets to a deeper level and extract interesting phenotype clusters for more comprehensive evaluation in an unbiased fashion upon application of this approach. Organelle-related protein clusters were identified beyond those sufficiently annotated for use as training data. Furthermore, we propose avenues for the incorporation of observations made into general practice for the classification of protein-organelle membership from quantitative MS experiments. BIOLOGICAL SIGNIFICANCE: Protein sub-cellular localisation plays an important role in molecular interactions, signalling and transport mechanisms. The prediction of protein localisation by quantitative mass-spectrometry (MS) proteomics is a growing field and an important endeavour in improving protein annotation. Several such approaches use gradient-based separation of cellular organelle content to measure relative protein abundance across distinct gradient fractions. The distribution profiles are commonly mapped in silico to known protein-organelle associations via supervised machine learning algorithms, to create classifiers that associate unannotated proteins to specific organelles. These strategies are prone to error, however, if organelle-related groupings present in experimental output are not represented, for example owing to the lack of existing annotation, when creating the protein-organelle mapping. Here, the application of a phenotype discovery approach to LOPIT gradient-based MS data identifies candidate organelle phenotypes for further evaluation in an unbiased fashion. Software implementation and usage guidelines are provided for application to wider protein-organelle association experiments. In the wider context, semi-supervised organelle discovery is discussed as a paradigm with which to generate new protein annotations from MS-based organelle proteomics experiments.

Method for suppressing non-specific protein interactions observed with affinity resins.

Previous high throughput data analysis from several different approaches to affinity purification of protein complexes have revealed catalogues of contaminating proteins that persistently co-purify. Some of these contaminating proteins appear to be specific to one particular affinity matrix used or even to the artificial affinity tags introduced into endogenous proteins for the purpose of purification. A recent approach to minimising non-specific protein interactions in high throughput screens utilises pre-equilibration of affinity surfaces with thiocyanate anions to reduce non-specific binding of proteins. This approach not only reduces the effect of contaminating proteins but also promotes the enrichment of the specific binding partners. Here, we have taken this method and adapted it in an attempt to reduce the abundance of common contaminants in affinity purification experiments. We found the effect varied depending on the bait used, most likely due to its endogenous abundance.

Proteomics of cryoprotective dehydration in Megaphorura arctica Tullberg 1876 (Onychiuridae: Collembola).

The Arctic springtail, Megaphorura arctica Tullberg 1876 (Onychiuridae: Collembola), is one of the few organisms known to survive the extreme stresses of its environment by using cryoprotective dehydration. We have undertaken a proteomics study comparing M. arctica, acclimated at -2°C, the temperature known to induce the production of the anhydroprotectant trehalose in this species, and -6°C, the temperature at which trehalose expression plateaus, against control animals acclimated at +5°C. Using difference gel electrophoresis, and liquid chromatography tandem mass spectrometry, we identified three categories of differentially expressed proteins with specific functions, up-regulated in both the -2°C and -6°C animals, that were involved in metabolism, membrane transport and protein folding. Proteins involved in cytoskeleton organisation were only up-regulated in the -6°C animals.

Biofilms and type III secretion are not mutually exclusive in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes acute and chronic infections in immunocompromised individuals. It is also a model organism for bacterial biofilm formation. Acute infections are often associated with planktonic or free-floating cells, high virulence and fast growth. Conversely, chronic infections are often associated with the biofilm mode of growth, low virulence and slow growth that resembles that of planktonic cells in stationary phase. Biofilm formation and type III secretion have been shown to be reciprocally regulated, and it has been suggested that factors related to acute infection may be incompatible with biofilm formation. In a previous proteomic study of the interrelationships between planktonic cells, colonies and continuously grown biofilms, we showed that biofilms under the growth conditions applied are more similar to planktonic cells in exponential phase than to those in stationary phase. In the current study, we investigated how these conditions influence the production of virulence factors using a transcriptomic approach. Our results show that biofilms express the type III secretion system, whereas planktonic cells do not. This was confirmed by the detection of PcrV in the cellular and secreted fractions of biofilms, but not in those of planktonic cells. We also detected the type III effector proteins ExoS and ExoT in the biofilm effluent, but not in the supernatants of planktonic cells. Biofilm formation and type III secretion are therefore not mutually exclusive in P. aeruginosa, and biofilms could play a more active role in virulence than previously thought.

Independent protein-profiling studies show a decrease in apolipoprotein A1 levels in schizophrenia CSF, brain and peripheral tissues.

Although some insights into the etiology of schizophrenia have been gained, an understanding of the illness at the molecular level remains elusive. Recent advances in proteomic profiling offer great promise for the discovery of markers underlying pathophysiology of diseases. In the present study, we employed two high-throughput proteomic techniques together with traditional methods to investigate cerebrospinal fluid (CSF), brain and peripheral tissues (liver, red blood cells and serum) of schizophrenia patients in an attempt to identify peripheral/surrogate disease markers. The cohorts used to investigate each tissue were largely independent, although some CSF and serum samples were collected from the same patient. To address the major confounding factor of antipsychotic drug treatment, we also included a large cohort of first-onset drug-naive patients. Apolipoprotein A1 (apoA1) showed a significant decrease in expression in schizophrenia patients compared to controls in all five tissues examined. Specifically, using SELDI-TOF mass spectrometry, apoA1 was found decreased in CSF from schizophrenia patients (-35%, P=0.00001) and, using 2D-DIGE, apoA1 was also found downregulated in liver (-30%, P=0.02) and RBCs (-60%, P=0.003). Furthermore, we found a significant reduction of apoA1 in sera of first-onset drug-naive schizophrenia patients using enzyme-linked immunosorbent assay (-18%, P=0.00008) and in two investigations of post-mortem brain tissue using western blot analysis (-35%, P=0.05; -51%, P=0.05). These results show that apoA1 is consistently downregulated in the central nervous system as well as peripheral tissues of schizophrenia patients and may be linked to the underlying disease mechanism.

Interrelationships between colonies, biofilms, and planktonic cells of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a gram-negative bacterium and an opportunistic human pathogen that causes chronic infections in immunocompromised individuals. These infections are hard to treat, partly due to the high intrinsic resistance of the bacterium to clinically used antibiotics and partly due to the formation of antibiotic-tolerant biofilms. The three most common ways of growing bacteria in vitro are as planktonic cultures, colonies on agar plates, and biofilms in continuous-flow systems. Biofilms are known to express genes different from those of planktonic cells, and biofilm cells are generally believed to closely resemble planktonic cells in stationary phase. However, few, if any, studies have examined global gene expression in colonies. We used a proteomic approach to investigate the interrelationships between planktonic cells, colonies, and biofilms under comparable conditions. Our results show that protein profiles in colonies resemble those of planktonic cells. Furthermore, contrary to what has been reported previously, the protein profiles of biofilms were found to more closely resemble those of exponentially growing planktonic cells than those of planktonic cells in the stationary phase. These findings raise some intriguing questions about the true nature of biofilms.

Cement from nanocrystalline hydroxyapatite: effect of calcium phosphate ratio.

Nanocrystalline hydroxyapatite (nHA) can be mixed with phosphoric acid to form a brushite cement; a degradable inorganic bone filling material. nHA was precipitated from reactants of calcium to phosphate (Ca/P) ratio 0.8 to 2.0 and mixed with phosphoric acid, which resulted in the formation of a brushite cement. Cement was also formed by mixing microcrystalline calcium phosphates, beta-tricalcium phosphate, hydroxyapatite and tetracalcium phosphate with phosphoric acid solution. Cement produced with nHA was stronger in compression than that formed with crystalline calcium phosphate phases. Setting time, strength and composition of cement produced with nHA was dependant on both the Ca/P ratio of nHA and the concentration of phosphoric acid in cement slurry. Increasing phosphoric acid concentration increased compressive strength whilst reducing the initial setting time of cement. Reducing the Ca/P ratio of nHA precipitation reactants retarded the setting and increased the extent of reaction of cements. This finding was unexpected and suggests that Ca/P ratio may strongly affect dissolution behaviour and this parameter is more important than stoichiometry in determining extent of reaction in this system. This study demonstrated that the wide variation in stoichiometry that may be attained in nanocrystalline apatite may be utilised to change cement performance and setting behaviour.

Cement from magnesium substituted hydroxyapatite.

Brushite cement may be used as a bone graft material and is more soluble than apatite in physiological conditions. Consequently it is considerably more resorbable in vivo than apatite forming cements. Brushite cement formation has previously been reported by our group following the mixture of nanocrystalline hydroxyapatite and phosphoric acid. In this study, brushite cement was formed from the reaction of nanocrystalline magnesium-substituted hydroxyapatite with phosphoric acid in an attempt to produce a magnesium substituted brushite cement. The presence of magnesium was shown to have a strong effect on cement composition and strength. Additionally the presence of magnesium in brushite cement was found to reduce the extent of brushite hydrolysis resulting in the formation of HA. By incorporating magnesium ions in the apatite reactant structure the concentration of magnesium ions in the liquid phase of the cement was controlled by the dissolution rate of the apatite. This approach may be used to supply other ions to cement systems during setting as a means to manipulate the clinical performance and characteristics of brushite cements.

Survival of Escherichia coli O157:H7, O111:H- and O26:H11 in artificially contaminated chocolate and confectionery products.

To date, the survival of Escherichia coli O157:H7 and other verocytotoxin-producing E. coli (VTEC) in chocolate and other confectionery products has not been fully established, unlike Salmonella, which have been responsible for occasional outbreaks of infection linked to contaminated chocolate and related products, although none of these outbreaks have been related to products produced in the United Kingdom. The United Kingdom Biscuit, Cake, Chocolate and Confectionery Alliance commissioned this study to obtain information on the decline and potential survival of E. coli, particularly verocytotoxin-producing strains, in reduced aw confectionery products chocolate, biscuit cream and mallow. These products were artificially contaminated with high (4 log10 cfu/g) and low (2 log10 cfu/g) levels of E. coli O157:H7, O111:H- and O26:H11 and their survival, as affected by storage temperature (10, 22 and 38 degrees C), was monitored over 12 months. Preliminary studies to establish the best inoculation and recovery procedures indicated that differences between counts on selective and non-selective media used were not sufficiently different to influence the outcome of this study. Irrespective of sample type, rapid decline was observed in products stored at 38 degrees C and increased survival occurred in products stored at 10 degrees C. In chocolate (average aw 0.40), these bacteria were detected for up to 43 days in samples stored at 38 degrees C. At 22 degrees C they survived for up to 90 days and in product stored at 10 degrees C they could still be detected after 366 days storage. In biscuit cream (average aw 0.75) they survived for 2 days at 38 degrees C, 42 days at 22 degrees C and 58 days at 10 degrees C. Whilst mallow (aw ca. 0.73) was not stored at 38 degrees C, these bacteria could still be detected in samples stored for up to 113 and 273 days at 22 and 10 degrees C, respectively. The observed prolonged survival of these bacteria under conditions of reduced aw and lowered storage temperature in this study is supported by previous studies with Salmonella and E. coli O157:H7 in other foods. In the same way that Salmonella bacteria can survive for long periods, in excess of 12 months, in chocolate, this study provides evidence that E. coli, including pathogenic strains, can also survive for similar periods of time. Assuming the routes of transmission are similar, controls currently used by the confectionery industry to prevent contamination by Salmonella should also be effective against E. coli, including VT-producing strains, providing that all raw materials have been suitably processed, stored and handled before and during manufacture.

Cements from nanocrystalline hydroxyapatite.

Calcium phosphate cements are used as bone substitute materials because they may be moulded to fill a void or defect in bone and are osteoconductive. Although apatite cements are stronger than brushite cements, they are potentially less resorbable in vivo. Brushite cements are three-component systems whereby phosphate ions and water react with a soluble calcium phosphate to form brushite (CaHPO4 x 2H2O). Previously reported brushite cement formulations set following the mixture of a calcium phosphate, such as beta-tricalcium phosphate (beta-TCP), with an acidic component such as H3PO4 or monocalcium phosphate monohydrate (MCPM). Due to its low solubility, hydroxyapatite (HA) is yet to be reported as a reactive component in calcium phosphate cement systems. Here we report a new cement system setting to form a matrix consisting predominantly of brushite following the mixture of phosphoric acid with nanocrystalline HA. As a result of the relative ease with which ionic substitutions may be made in apatite this route may offer a novel way to control cement composition or setting characteristics. Since kinetic solubility is dependent on particle size and precipitation temperature is known to affect precipitated HA crystal size, the phase composition and mechanical properties of cements made from HA precipitated at temperatures between 4 and 60 degrees C were investigated.

Localization of organelle proteins by isotope tagging (LOPIT).

We describe a proteomics method for determining the subcellular localization of membrane proteins. Organelles are partially separated using centrifugation through self-generating density gradients. Proteins from each organelle co-fractionate and therefore exhibit similar distributions in the gradient. Protein distributions can be determined through a series of pair-wise comparisons of gradient fractions, using cleavable ICAT to enable relative quantitation of protein levels by MS. The localization of novel proteins is determined using multivariate data analysis techniques to match their distributions to those of proteins that are known to reside in specific organelles. Using this approach, we have simultaneously demonstrated the localization of membrane proteins in both the endoplasmic reticulum and the Golgi apparatus in Arabidopsis. Localization of organelle proteins by isotope tagging is a new tool for high-throughput protein localization, which is applicable to a wide range of research areas such as the study of organelle function and protein trafficking.

The use of isotope-coded affinity tags (ICAT) to study organelle proteomes in Arabidopsis thaliana.

Organelle proteomics is the analysis of the protein contents of a subcellular compartment. Proteins identified in subcellular proteomic studies can only be assigned to an organelle if there are no contaminants present in the sample preparation. As a result, the majority of plant organelle proteomic studies have focused on the chloroplast and mitochondria, which can be isolated relatively easily. However, the isolation of components of the endomembrane system is far more difficult due to their similar sizes and densities. For this reason, quantitative proteomics methods are being developed to enable the assignment of proteins to a specific component of the endomembrane system without the need to obtain pure organelles.

Mitochondrial dysfunction in schizophrenia: evidence for compromised brain metabolism and oxidative stress.

The etiology and pathophysiology of schizophrenia remain unknown. A parallel transcriptomics, proteomics and metabolomics approach was employed on human brain tissue to explore the molecular disease signatures. Almost half the altered proteins identified by proteomics were associated with mitochondrial function and oxidative stress responses. This was mirrored by transcriptional and metabolite perturbations. Cluster analysis of transcriptional alterations showed that genes related to energy metabolism and oxidative stress differentiated almost 90% of schizophrenia patients from controls, while confounding drug effects could be ruled out. We propose that oxidative stress and the ensuing cellular adaptations are linked to the schizophrenia disease process and hope that this new disease concept may advance the approach to treatment, diagnosis and disease prevention of schizophrenia and related syndromes.

Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE).

Two-dimensional gel electrophoresis (2-D GE) is a key tool for comparative proteomics research. With its ability to separate complex protein mixtures with high resolution, 2-D GE is a technique commonly employed for protein profiling studies. Significant improvements have been made in 2-D GE technology with the development of two-dimensional fluorescence difference gel electrophoresis (2-D DIGE), where proteins are first labelled with one of three spectrally resolvable fluorescent cyanine dyes before being separated over first and second dimensions according to their charge and size, respectively. When used in conjunction with automated analysis packages, this multiplexing approach can accurately and reproducibly quantify protein expression for control and experimental groups. Differentially expressed proteins can be subsequently identified by mass spectrometric methods. Here, we describe the successful application and optimisation of 2-D DIGE technology for human postmortem brain studies. This technology, especially when coupled with other functional genomics approaches, such as transcriptomics and metabolomics studies, will enhance our current understanding of human disease and lead to new therapeutic and diagnostic possibilities.

Phosphorylation of elongation factor-2 kinase on serine 499 by cAMP-dependent protein kinase induces Ca2+/calmodulin-independent activity.

Elongation factor-2 kinase (eEF-2K) negatively regulates mRNA translation via the phosphorylation and inactivation of elongation factor-2 (eEF-2). We have shown previously that purified eEF-2K can be phosphorylated in vitro by cAMP-dependent protein kinase (PKA) and that this induces significant Ca(2+)/calmodulin (CaM)-independent eEF-2K activity [Redpath and Proud (1993) Biochem. J. 293, 31-34]. Furthermore, elevation of cAMP levels in adipocytes also increases the level of Ca(2+)/CaM-independent eEF-2K activity to a similar extent, providing a mechanistic link between elevated cAMP and the inhibition of protein synthesis [Diggle, Redpath, Heesom and Denton (1998) Biochem. J. 336, 525-529]. Here we describe the expression of glutathione S-transferase (GST)-eEF-2K fusion protein and the identification of two serine residues that are phosphorylated by PKA in vitro. Endoproteinase Arg-C digestion of GST-eEF-2K produced two phosphopeptides that were separated by HPLC and sequenced. (32)P Radioactivity release from these peptides indicated that the sites of phosphorylation were Ser-365 and Ser-499, both of which lie C-terminal to the catalytic domain. Mutation of these sites to non-phosphorylatable residues indicated that both sites need to be phosphorylated to induce Ca(2+)/CaM-independent eEF-2K activity in vitro. However, expression of Myc-tagged eEF-2K in HEK 293 cells, followed by treatment with chlorophenylthio-cAMP (CPT-cAMP), showed that Ser-499 phosphorylation alone induced Ca(2+)/CaM-independent eEF-2K activity in cells. Co-expression of wild-type eEF-2K with luciferase resulted in a 2-3-fold reduction in luciferase expression. Expression of eEF-2K S499D resulted in a 10-fold reduction in luciferase expression despite the fact that this mutant was expressed at very low levels. This indicates that eEF-2K S499D is constitutively active when expressed in cells, thus leading to the suppression of its own expression. Our data demonstrate an important role for the phosphorylation of Ser-499 in the activation of eEF-2K by PKA and the inhibition of protein synthesis.

Survey of current trends in DNA synthesis core facilities.

The Nucleic Acids Research Group of the Association of Biomolecular Resource Facilities (ABRF) last surveyed DNA synthesis core facilities in April 1995. Because of the introduction of new technologies and dramatic changes in the market, we sought to update survey information and to determine how academic facilities responded to the challenge presented by commercial counterparts. The online survey was opened in January 1999 by notifying members and subscribers to the ABRF electronic discussion group. The survey consisted of five parts: general facility information, oligonucleotide production profile, oligonucleotide charges, synthesis protocols, and trends in DNA synthesis (including individual comments). All submitted data were anonymously coded. Respondents from DNA synthesis facilities were primarily from the academic category and were established between 1984 and 1991. Typically, a facility provides additional services such as DNA sequencing and has upgraded to electronic ordering. There is stability in staffing profiles for these facilities in that the total number of employees is relatively unchanged, the tenure for staff averages 5.9 years, and experience is extensive. On average, academic facilities annually produce approximately 1/16 the number of oligonucleotides produced by the average commercial facilities, but all facilities report an increase in demand. Charges for standard oligonucleotides from academic facilities are relatively higher than from commercial companies; however, the opposite is true for modified phosphoramidites. Subsidized facilities charge less than nonsubsidized facilities. Synthesis protocols and reagents are standard across the categories. Most facilities offer typical modifications such as biotinylation. Despite the competition by large commercial facilities that have reduced costs dramatically, academic facilities remain a stable entity. Academic facilities enhance the quality of service by focusing on nonstandard oligonucleotides and valuable services such as personal consultations, electronic ordering, and diversifying into other services.

Capillary electrophoresis with electrospray mass spectrometry detection for low-molecular-mass compounds.

Mass spectrometry (MS) detection using electrospray ionization (ESI) has been explored for the separation by capillary electrophoresis (CE) of a number of sample mixtures containing low-molecular-mass species. Optimal sheath liquid composition has been determined using a peptide mixture in which femtomolar quantities of analyte were easily observed. Effects of CE buffer choice were studied in detail. Also, a separation of basic drugs in cough syrup has been successfully detected by ESI-MS. Using negative ionization, a mixture of alkyl sulfonates and a mixture of food dyes were analyzed. All components were easily resolved and identified by molecular weight.