Biomedical subjects

Karin E Markides

Publications and source records attributed to Karin E Markides.

At least 37 records · Page 2Linked to original sources

PubMed2004

A chemometric study of active parameters and their interaction effects in a nebulized sheath-liquid electrospray interface for capillary electrophoresis-mass spectrometry.

A chemometrics approach has been used for evaluating the effect of four experimental parameters when coupling capillary electrophoresis (CE) to electrospray ionization-mass spectrometry (ESI-MS). Electrospray voltage, sheath-liquid flow rate, nebulizing gas flow rate, and spray needle position in respect to the MS orifice were varied according to a full factorial design. In addition to main effects, two interaction effects could be identified as significant when measuring the peak intensity of the analytes, from a sample mixture containing peptides and pharmaceuticals. The first interaction effects, between the nebulizing gas flow rate and the sheath-liquid flow rate, and the second interaction effect, between the nebulizing gas flow rate and the spray position, could further explain the impact that these variables have on the spray performance. The number of theoretical plates and the baseline noise were also measured. The sheath-liquid flow was found to significantly affect the separation efficiency, while the noise level mainly was controlled by the nebulizing gas flow. The same factorial design was also used for a CE capillary with lower internal diameter (ID) and the effects of the same variables were compared on those capillaries using equal injection volume for both capillaries. Similar trends were obtained in both capillaries but capillary ID was shown to be a significant variable when evaluating both capillaries in a single model. It was found that a capillary with 25 microm ID provided improved CE-MS performance over than corresponding 50 microm ID capillary. Enhanced sensitivity was obtained using the narrow-bore capillary, and at lower sheath-liquid flow rate the 25 microm ID capillary also gave rise to more efficient peaks.

Electrophoresis, Capillary↗

PubMed2004

Cerebrospinal fluid protein patterns in neurodegenerative disease revealed by liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry.

This study demonstrates the power of a novel proteomic approach developed for the detection and identification of biological markers in body fluids. The goal was to observe alterations in the protein patterns of cerebrospinal fluid (CSF) related to amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder with unknown etiology. In the experiments, tryptic digests of CSF from patients and healthy controls were analyzed by on-line capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry. (FT-ICR MS) Typically, around 4000 peptides were detected in one such experiment, and a pattern recognition program was constructed for the data analysis to distinguish mass chromatograms from patients and controls. This strategy was evaluated comparing the peptide patterns of CSF spiked in vitro with a biomarker, with control CSF. The patterns were clearly separated and the tryptic peptides of the biomarker were successfully selected as characteristic peaks. Hence, the method was applied to compare mass chromatograms of CSF from 12 ALS-patients and 10 matched healthy controls. While no biomarker alone could be identified from the characteristic peaks, we were able to assign 4 out of 5 unknown samples correctly (i.e., 80% correctly diagnosed, 20% false-negative), and it would be 100% if we reject a possible outlier believed to be caused by an occlusion in the spinal CSF compartment. These findings are very promising, although the clinical relevance is not fully established due to the low number of unknown samples analyzed. In addition to the diagnostic potential, these results may be important steps towards understanding the neurodegenerative process.

Adult↗

PubMed2004

Capillary electrophoresis off-line matrix-assisted laser desorption/ionisation mass spectrometry of intact and digested proteins using cationic-coated capillaries.

Capillary electrophoresis (CE) was coupled off-line with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analysis of proteins and peptides. CE fractions were collected directly on a matrix-coated MALDI target, using a sheath-flow interface. Protein adsorption during CE separations was prevented by coating the capillaries with the physically adsorbed, cationic polymer PolyE-323. The CE/MALDI-MS system was used for the analysis of model proteins and peptides at physiological pH as well as analysis of proteins in tear fluid. Moreover, tryptic on-target digestion of the collected protein fractions, with subsequent MALDI-MS and MS/MS peptide analysis, was demonstrated.

Electrophoresis, Capillary↗

PubMed2003

Novel approach to the determination of structurally similar hydroxamate siderophores by column-switching capillary liquid chromatography coupled to mass spectrometry.

In this study a new approach to determine three different siderophores (ferrichrome, ferrichrysin, ferricrocin) in natural soil solutions as well as in cultures of fungi is presented. The method includes enrichment of the analytes on a short pre-column, packed with C18 material, and subsequent highly selective separation of the analytes on a capillary porous graphitic carbon (PGC) column. In contrast to normal C18 packing materials, porous graphitic carbon offers chromatographic resolution between the three very similar analytes. The selectivity of the method is enhanced even further by the electrospray ionization (ESI) mass spectrometric detection. The combination of a short pre-column and a packed capillary separation column results in a method with high sensitivity. Reported detection limits, defined as the concentration giving the signal-to-noise ratio 3:1, is 27.7 pM for ferrichrome, 46.1 pM for ferricrocin and 37.4 pM for ferrichrysin.

Chromatography, Liquid↗

PubMed2003

Development of a poly(dimethylsiloxane) interface for on-line capillary column liquid chromatography-capillary electrophoresis coupled to sheathless electrospray ionization time-of-flight mass spectrometry.

An interface in elastomeric poly(dimethylsiloxane) (PDMS) for on-line orthogonal coupling of packed capillary liquid chromatography (LC) (i.d. = 0.2 mm) with capillary electrophoresis (CE) in combination with sheathless electrospray ionization (ESI) time-of-flight mass spectrometric (TOFMS) detection is presented. The new interface has a two-level design, which in combination with a continuous CE electrolyte flow through the interface provides integrity of the LC effluent and the CE separation until an injection is desired. The transparent and flexible PDMS material was found to have a number of advantages when combined with fused silica column technology, including ease to follow the process and ease to exchange columns. By combining conventional microscale systems of LC, CE, and ESI-MS, respectively, the time scales of the individual dimensions were harmonized for optimal peak capacity per unit time. The performance of the LC-CE-TOFMS system was evaluated using peptides as model substances. A S/N of about 330 was achieved for leucine-enkephaline from a 0.5 microL LC injection of 25 microg/mL peptide standard.

Journal Article↗

PubMed2003

Analysis of phosphatidylcholine and sphingomyelin molecular species from brain extracts using capillary liquid chromatography electrospray ionization mass spectrometry.

One feature of complex lipids is that many subtypes of these molecules exist as a diverse mixture in a biological sample. Qualitative and quantitative analysis of these closely related molecules require sensitive and specific analytical methods to detect intact phospholipids (PL) and sphingomyelin (SM) species and to differentiate between them. Conventional analytical methods require laborious procedures including separation by column, argentation thin-layer chromatography or liquid chromatography (LC) after pre- or post-column derivatization. In the present work, a method based on reversed phase capillary LC coupled on-line to electrospray ionization mass spectrometry (LC/ESI/MS) has been developed to gather tools for lipidomic studies, i.e. the profiling of complex mixtures of lipids in small amounts of various cells and tissues. The LC/MS system used consisted of an LC pump in an isocratic elution, a reversed phase capillary column and a single quadrupole mass spectrometer operated in the positive ion mode. A successful separation of phosphatidylcholine (PC) and SM molecular species was obtained with a minimum detectable quantity (MDQ) in the low fmol range injected on column. The method was applied to human brain extracts. Furthermore, the extraction efficiencies of the traditional Folch method and pressurized fluid extraction (PFE) were compared using the human brain. It was found that the intensity of the PC and SM molecular species extracted by PFE is two times that of Folch.

Aged↗

PubMed2003

Novel polyamine coating providing non-covalent deactivation and reversed electroosmotic flow of fused-silica capillaries for capillary electrophoresis.

A new polycationic coating for use in capillary electrophoresis has been developed that enables chemical modification of fused-silica capillary surfaces for analysis of compounds like basic proteins. The cationic polyamine, containing short aliphatic blocks of combined 2 and 3-carbon length, was physically adsorbed onto the negatively charged fused-silica surface through ionic interaction by flushing the capillary with a polyamine solution, followed by a self-stabilization step. The polyamine coated capillaries generated an anodal electroosmotic flow that was independent of pH in the investigated range of pH 4-8. The capillary performance was demonstrated by fast separations of basic proteins with peak efficiencies in the range of 265,000-584,000 plates.

Adsorption↗

PubMed2003

Miniaturized on-line proteolysis-capillary liquid chromatography-mass spectrometry for peptide mapping of lactate dehydrogenase.

In this study, methodology was developed for on-line and miniaturized enzymatic digestion with liquid chromatographic (LC) separation and mass spectrometric (MS) detection. A packed capillary LC-MS system was combined with on-line trypsin cleavage of a model protein, lactate dehydrogenase, to provide an efficient system for peptide mapping. The protein was injected onto an enzymatic capillary reactor and the resulting peptides were efficiently trapped on a capillary trapping column. Different trapping columns were evaluated to achieve a high binding capacity for the peptides generated in the enzyme reactor. The peptides were further eluted from the pre-column and separated on an analytical capillary column by a buffer more suitable for the following an electrospray ionisation (ESI) MS process. An important aspect of the on-line approach was the desalting of peptides performed in the trapping column to avoid detrimental signal suppression in the ESI process. The developed on-line system was finally compared to a classical digestion in solution, with reference to peptide sequence coverage and sensitivity. It was shown that the on-line system gave more than 100% higher peptide sequence coverage than traditional digestion methods.

Animals↗

PubMed2003

On-column polymer-imbedded graphite inlet electrode for capillary electrophoresis coupled on-line with flow injection analysis in a poly(dimethylsiloxane) interface.

A method for coupling an electrophoretic driven separation to a liquid flow, using conventional fused-silica capillaries and a soft polymeric interface is presented. A novel design of the electrode providing high voltage to the electrophoretic separation was also developed. The electrode consisted of a conductive polyimide/graphite imbedded coating immobilized onto the capillary electrophoresis (CE) column inlet. This integrated electrode gave the same separation performance as a commonly used platinum electrode. The on-column electrode also showed good electrochemical stability in chronoamperometric experiments. In addition, with this electrode design, the electrode position relative to the inlet end of the CE column will always be constant and well defined. The on-line flow injection analysis (FIA)-CE system was used with electrospray ionization (ESI)-time of flight (TOF)-mass spectrometry detection. The preparation of the PDMS (poly(dimethylsiloxane)) interface for FIA-CE is described in detail and used for initial tests of the on-column polymer-imbedded graphite inlet electrode. In this interface, a pressure-driven liquid flow, a make up CE electrolyte and a CE column inlet meet in a two-level cross (95 microm ID) in the PDMS structure, enabling independent flow characterization.

Dimethylpolysiloxanes↗

PubMed2003

Protein identification in cerebrospinal fluid using packed capillary liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry.

The identification and characterization of proteins in complex biological samples such as body fluids, require powerful and reliable tools. Mass spectrometry is today one of the most important methods in such research. This paper reports on the results from the first experiment where a tryptic digest of cerebrospinal fluid was analyzed applying reversed phase liquid chromatography coupled on-line to a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer. In total, 70 204 peaks were detected, which originated from 16 296 isotopic clusters corresponding to 6551 unique peptide masses. From these masses, 39 proteins were identified in the sample. The amount of sample required for one experiment corresponds to 32 microL of cerebrospinal fluid.

Biophysics↗

PubMed2003

Classification of lactate dehydrogenase of different origin by liquid chromatography-mass spectrometry and multivariate analysis.

A method intended to serve as a multivariate quality control tool in the production of pharmaceutical proteins is presented. The method is based on multivariate analysis of peptide maps generated with liquid chromatography-mass spectrometry (LC-MS). Lactate dehydrogenase (LDH) from different species and tissues were used as model compounds in the study. The proteins were digested with Endoproteinase Lys-C before the LC-MS analysis. After data pretreatment of the peptide maps, successful classification of the LDHs were obtained by discriminant analysis with partial least squares regression and artificial neural networks. Further, principal component analysis was applied to visualize the relationships between the samples.

Animals↗

PubMed2003

A comparison of the electrochemical stabilities of metal, polymer and graphite coated nanospray emitters.

Chronoamperometry (CA) and cyclic voltammetry (CV) were used to compare the electrochemical behavior of metal, polymer and graphite coated nanospray emitters. It is shown that electrochemical reactions occurring at the emitter surface limit the lifetime of the noble metal coated nanospray emitters while the graphite coated nanospray emitters show good electrochemical stabilities. Although the surface of the graphite coated emitters may be passivated at positive potentials, the conductive coating is not lost as for the noble metal coated nanospray emitters. The graphite coated nanospray emitters still produced a stable nanospray signal despite the presence of a passivated surface. The polymer (i.e. polyaniline) coated nanospray emitters showed very low electrochemical activity and could not be thoroughly tested by CA. The relative short lifetimes seen in the electrochemical tests are qualitatively comparable with those obtained in nanospray experiments, in which only the outmost tip of the emitter is electrochemically active. However, the electrochemical stress during CA far exceeds the stress during ESI, which implies that CA can be used to perform quick and simple estimates of emitter stabilities. To our knowledge, this is the first time the electrochemical behavior of metal, polymer and graphite coated nanospray emitters has been compared.

Journal Article↗

PubMed2003

A novel mass spectrometric approach to the analysis of hormonal peptides in extracts of mouse pancreatic islets.

Liquid chromatography mass spectrometry (LC-MS) is a valuable tool in the analysis of proteins and peptides. The combination of LC-MS with different fragmentation methods provides sequence information on components in complex mixtures. In this work, on-line packed capillary LC electrospray ionization Fourier transform ion cyclotron resonance MS was combined with two complementary fragmentation techniques, i.e. nozzle-skimmer fragmentation and electron capture dissociation, for the determination of hormonal peptides in an acid ethanol extract of mouse pancreatic islets. The most abundant peptides, those derived from proinsulin and proglucagon, were identified by their masses and additional sequence-tag information established their identities. Interestingly, the experiments demonstrated the presence of truncated C-peptides, des-(25-29)-C-peptide and des-(27-31)-C-peptide. These novel findings clearly illustrate the potential usefulness of the described technique for on-line sequencing and characterization of peptides in tissue extracts.

Animals↗

PubMed2002

Copolymeric (lR-trans)-N,N'-1,2-cyclohexylene-bisbenzamide oligodimethylsiloxane chiral stationary phase for gas chromatography.

A copolymeric stationary phase, consisting of a chiral selective part, i.e. (1R-trans)-N,N'-1,2-cyclohexylenebisbenzamide, and an efficient siloxane oligomeric part, was successfully applied to open tubular column GC analysis. The efficiency and the chiral selectivity of this stationary phase were studied in detail, and high capacity and efficiency at elevated GC temperatures were especially noted. Several drugs and other enantiomeric pairs were separated. The shown examples demonstrate a broad application range for this type of chiral stationary phase in GC analysis.

Benzamides↗

PubMed2002

Quantitation of tryptophan, kynurenine and kynurenic acid in human plasma by capillary liquid chromatography-electrospray ionization tandem mass spectrometry.

Concentrations of tryptophan and its metabolites in plasma are of great interest in determining proper diagnosis and medication of several neurological diseases like, for example, Alzheimer's disease. A method of standard addition was developed to determine total level of tryptophan and two of its metabolites, kynurenine and kynurenic acid, in human plasma by capillary liquid chromatography-electrospray ionization tandem mass spectrometry. Plasma samples were simply deproteinized by addition of diluted perchloric acid. Samples were then mixed with trichloroacetic acid and injected onto a capillary column. Analytes were separated by a fast gradient elution of the injected samples. Detection was performed by sheathless electrospray tandem mass spectrometry in the multiple reaction monitoring mode. Linear calibration curves were obtained for spiked plasma sample with up to 100% of the expected analytes concentrations. The determined concentrations were well within ranges previously reported (i.e., 6 nM-95 microM) and limit of detections were around 3 nM for each analyte.

Calibration↗

PubMed2002

Prediction of chromatographic retention and protein identification in liquid chromatography/mass spectrometry.

Liquid chromatography coupled on- or off-line with mass spectrometry is rapidly advancing as a tool in proteomics capable of dealing with the inherent complexity in biology and complementing conventional approaches based on two-dimensional gel electrophoresis. Proteins can be identified by proteolytic digestion and peptide mass fingerprinting or by searching databases using short-sequence tags generated by tandem mass spectrometry. This paper shows that information on the chromatographic behavior of peptides can assist protein identification by peptide mass fingerprinting in liquid chromatography/mass spectrometry. This additional information is significant and already available at no extra experimental cost.

Amino Acids↗

PubMed2002

Determination of boron-containing compounds in urine and blood plasma from boron neutron capture therapy patients. The importance of using coupled techniques.

The necessity of using coupled techniques to analyze samples from boron neutron capture therapy (BNCT) patients prior to element-specific detection has been demonstrated. BNCT patients were infused with p-boronophenylalanine (BPA)-fructose complex before the therapy started. Urine and blood plasma samples were collected at different times after the start of the BPA administration and were run on a porous graphitic carbon column coupled on-line to an inductively coupled plasma-atomic emission spectrometer (ICP-AES) and an ICP time-of-flight mass spectrometer (TOF-MS). In addition to BPA, a possible metabolite to BPA and some minor boron-containing compounds, eluting close to the front, were also found in the urine and plasma samples. Because only the total concentration of boron has been measured so far in earlier studies, the suspected metabolite could not be detected, and this is the first report indicating its presence in urine and plasma of BNCT patients. The abundance of 10B in urine was about the same for BPA and its possible metabolite (98-99%). The ratio between the possible metabolite and BPA was found to differ in the urine from different patients. Most of the patients had a metabolite concentration of approximately 10 mol % of the BPA content in their urine 5-11 h after the start of the BPA administration. This ratio increased to between 30 and 80% when 24 h had passed. The ratio of metabolite to BPA was found to be lower in the plasma than in the urine samples at comparable time after the start of BPA infusion. Preliminary results from micro-LC-electrospray ionization (ESI)-MS/MS measurements on four urine samples indicate that the metabolite has a higher mass than BPA.

Boron Compounds↗

PubMed2002

On-line coupling of microdialysis to packed capillary column liquid chromatography-tandem mass spectrometry demonstrated by measurement of free concentrations of ropivacaine and metabolite from spiked plasma samples.

An on-line coupling of microdialysis to a packed capillary column switching liquid chromatographic system (0.2 mm I.D.) and mass spectrometric detection was developed. The microdialysates were collected in the loop of the first of three valves, coupled in direct series. A deuterated internal standard was added on-line by the middle valve and the third valve operated both a pre-column, for desalting of the physiological buffer used in the sampling procedure, and a separation column. The on-line system was used to study free concentrations of ropivacaine and its metabolite (PPX) in human plasma samples. The analytes were detected by electrospray ionization in a tandem mass spectrometer operating in multiple reaction monitoring mode. The free fractions of ropivacaine (200 nM total concentration) and PPX (20 nM total concentration) in spiked plasma samples were 12 +/- 3 and 47 +/- 5% (+/- standard deviation for day-to-day variations, n=3), respectively.

Amides↗