Biomedical subjects

Barry L Karger

Publications and source records attributed to Barry L Karger.

32 records · Page 2Linked to original sources

PubMed2003

Closely spaced external standard: a universal method of achieving 5 ppm mass accuracy over the entire MALDI plate in axial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Close deposition of the sample and external standard was used in axial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to achieve mass accuracy equivalent to that obtained with an internal standard across the entire MALDI plate. In this work, the sample and external standard were deposited by continuous deposition in separate traces, each approximately 200 micro m wide. The dependence of the mass accuracy on the distance between the sample and standard traces was determined across a MALDI target plate with dimensions of 57.5 mm x 57.0 mm by varying the gap between the traces from 100 micro m to 4 mm. During acquisition, two adjacent traces were alternately irradiated with a 200-Hz laser, such that the peaks in the resulting mass spectra combined the sample and external standard. Ion suppression was not observed even when the peptide concentrations in the two traces differed by more than two orders of magnitude. The five peaks from the external standard trace were used in a four-term mass calibration of the masses of the sample trace. The average accuracy across the whole plate with this method was 5 ppm when peaks of the sample trace had signal-to-noise ratios of at least 30 and the gap between the traces was approximately 100 micro m. This approach was applied to determining peptide masses of a reversed-phase liquid chromatographic (LC) separation of a tryptic digest of beta-galactosidase deposited as a long serpentine trace across the MALDI plate, with accuracy comparable to that obtainable using internal calibration. In addition, the eluent from reversed-phase LC separation of a strong cation-exchange fraction containing tryptic peptides from a yeast lysate along with the closely placed external standard was deposited on the MALDI plate. The data obtained in the MS and MS/MS modes on a MALDI-TOF/TOF mass spectrometer were combined and used in database searching with MASCOT. Since the significant score is a function of mass accuracy in the MS mode, database searching with high mass accuracy reduced the number of false positives and also added peptides which otherwise would have been eliminated at lower mass accuracy (false negatives).

Calibration↗

PubMed2002

Multiple open-channel electroosmotic pumping system for microfluidic sample handling.

The development of a novel, fully integrated, miniaturized pumping system for generation of pressure-driven flow in microfluidic platforms is described. The micropump, based on electroosmotic pumping principles, has a multiple open-channel configuration consisting of hundreds of parallel, small-diameter microchannels. Specifically, pumps with microchannels of 1-6 microm in depth, 4-50 mm in length, and an overall area of a few square millimeters, were constructed. Flow rates of 10-400 nL/min were generated in electric-field-free regions in a stable, reproducible and controllable manner. In addition, eluent gradients were created by simultaneously using two pumps. Pressures up to 80 psi were produced with the present pump configurations. The pump can be easily interfaced with other operational elements of a micrototal analysis system (micro-TAS) device with multiplexing capabilities. A new microfluidic valving system was also briefly evaluated in conjunction with these pumps. The micropump was utilized to deliver peptide samples for electrospray ionization-mass spectrometric (ESI-MS) detection.

Electrochemistry↗

PubMed2002

Capillary array electrophoresis-MALDI mass spectrometry using a vacuum deposition interface.

We previously introduced a vacuum deposition interface for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF MS) on a moving surface (e.g., quartz wheel, Mylar tape, metal target). In our present work, the approach has been extended to demonstrate parallel analysis for multiple on-line infusion MALDI MS and capillary array electrophoresis (CAE)-MALDI MS. In the infusion mode, individual peptide samples were simultaneously deposited on a Mylar tape cartridge using an array of eight capillaries, yielding eight parallel traces. For CAE-MALDI/TOF MS, the same number of separation capillaries were coupled with an array of eight infusion capillaries using a common liquid junction, containing matrix solution. A fast-scanning mirror was employed to traverse the beam of the desorption laser across the Mylar tape to probe one trace at a time. The positions of the eight sample traces formed on the tape were automatically determined, and all samples were analyzed in rapid sequence using a kilohertz repetition rate laser and a high-throughput data acquisition system. The instrumentation was operated with CAE MS for high-throughput analysis without compromising data quality. The principles of parallel separation-vacuum deposition should be generally applicable to MALDI/TOF MS analysis for proteomics and other areas where separation and high throughput are required.

Angiotensins↗

PubMed2002

Design of a fraction collector for capillary array electrophoresis.

This paper describes a prototype instrument for high-throughput fraction collection with capillary array electrophoresis (CAE). The design of the system was based on a comprehensive collection approach, in which fractions from all capillaries were simultaneously collected in individual collection microwells in predefined time intervals. The location of the fractions in the microwells on the collection plate was determined by monitoring the individual zone velocities close to the end of each capillary. The collection microwell plate was fabricated from buffer-saturated agarose gel, which maintained permanent electrical contact with the separation capillaries during the collection process. Since the collection gel plate consisted of over 90% water, liquid evaporation from the collection wells was minimized. A 12-capillary array instrument was built with two-point detection using a side illumination scheme. The collection performance was demonstrated by reinjection of selected fractions of a double-stranded DNA (dsDNA) separation. The identity of collected DNA fragments was confirmed by PCR and sequencing.

DNA↗

PubMed2002

DNA sequencing with hydrophilic and hydrophobic polymers at elevated column temperatures.

Read length in DNA sequencing by capillary electrophoresis at elevated temperatures is shown to be greatly affected by the extent of hydrophobicity of the polymer separation matrix. At column temperatures of up to 80 degrees C, hydrophilic linear polyacrylamide (LPA) provides superior read length and separation speed compared to poly(N,N-dimethylacrylamide) (PDMA) and a 70:30 copolymer of N,N-dimethylacrylamide and N,N-diethylacrylamide (PDEA30). DNA-polymer and polymer intramolecular interactions are presumed to be a major cause of band broadening and the subsequent loss of separation efficiency with the more hydrophobic polymers at higher column temperatures. With LPA, these interactions were reduced, and a read length of 1000 bases at an optimum temperature of 70 degrees -75 degrees C was achieved in less than 59 min. By comparison, PDMA produced a read length of roughly 800 bases at 50 degrees C, which was close to the read length attained in LPA at the same temperature; however, the migration time was approximately 20% longer, mainly because of the higher polymer concentration required. At 60 degrees C, the maximum read length was 850 bases for PDMA, while at higher temperatures, read lengths for this polymer were substantially lower. With the copolymer DEA30, read length was 650 bases at the optimum temperature of 50 degrees C. Molecular masses of these polymers were determined by tandem gel permeation chromatography-multiangle laser light scattering method (GPC-MALLS). The results indicate that for long read, rapid DNA sequencing and analysis, hydrophilic polymers such as LPA provide the best overall performance.

Acrylamides↗

PubMed2002

DNA sequencing of close to 1000 bases in 40 minutes by capillary electrophoresis using dimethyl sulfoxide and urea as denaturants in replaceable linear polyacrylamide solutions.

The goal of this work was to reduce the capillary electrophoresis (CE) separation time of DNA sequencing fragments with linear polyacrylamide solutions while maintaining the previously achieved long read lengths of 1000 bases. Separation speed can be increased while maintaining long read lengths by reducing the separation matrix viscosity and/or raising the column temperature. As urea is a major contributor to the separation buffer viscosity, reducing its concentration is desirable both for increase in the separation speed and easier solution replacement from the capillary. However, at urea concentrations below 6 M, the denaturing capacity of the separation buffer is not sufficient for accurate base-calling. To restore the denaturing properties of the buffer, a small amount of an organic solvent was added to the formulation. We found that a mixture of 2 M urea with 5% v/w of dimethyl sulfoxide (DMSO) resulted in 975 bases being sequenced at 70 degrees C in 40 min with 98.5% accuracy. To achieve this result, the software was modified to perform base-calling at a peak resolution as low as 0.24. It is also demonstrated that the products of thermal decomposition of urea had a deleterious effect on the separation performance at temperatures above 70 degrees C. With total replacement of urea with DMSO, at a concentration of 5% v/w in the same linear polyacrylamide (LPA)-containing buffer, it was possible to increase the column temperature up to 90 degrees C. At this temperature, up to 951 bases with 98.5% accuracy could be read in only 32 min of separation. However, with DMSO alone, some groups of C-terminated peaks remained compressed, and column temperature at this level cannot at present be utilized with existing commercial instrumentation.

Acrylic Resins↗

PubMed2002

A miniaturized multichamber solution isoelectric focusing device for separation of protein digests.

A miniaturized multichamber device was constructed for solution isoelectric focusing (IEF) separation of complex peptide mixtures. The system, based on immobilized pH gels, consisted of 96 minichambers ( approximately 75 nuL each) arranged in eight rows. Neighboring chambers in a given row were separated by short glass tubes (4 mm inner diameter, 3 mm long), within which Immobiline gels of specific pH values were polymerized. During focusing, the device was sandwiched between two supporting blocks incorporating the reservoirs for anolyte and catholyte. In principle, multiple samples could be simultaneously fractionated, each separated into 12 fractions of various pI ranges. A variety of standard peptide mixtures and tryptic digests of proteins were separated by IEF using this device, and the fractions were characterized by mass spectrometry. For a codigested nine-protein mixture, both the total number of peptides identified and the average sequence coverage were similar to the results of ion-exchange chromatography (IEC), according to matrix assisted laser/desorption/ionization--time of flight (MALDI-TOF) data. The IEF separation provided concentrated and desalted fractions, suitable for an additional separation liquid chromatography, capillary electrophoresis (LC, CE) or mass spectrometry (MS) detection without additional sample cleanup. High loading capacity was achieved for the miniaturized multichamber IEF device. Importantly, a linear correlation was found between the experimentally determined and calculated pI values of peptides.

Acrylamides↗

PubMed2002

Analytical aspects of mass spectrometry and proteomics.

Mass spectrometry plays an essential role in proteomics analysis and research. In recent years, it has been increasingly recognized that a key to proteomics using mass spectrometry relies not only on the instrument itself, but also on the analytical strategies and front-end sample-handling techniques. The advances of separations and mass spectrometry are having an increasing impact on the discovery of disease biomarkers and the understanding of cellular processes.

Affinity Labels↗

PubMedDate not supplied

High-throughput microcoil NMR of compound libraries using zero-dispersion segmented flow analysis.

An automated system for loading samples into a microcoil NMR probe has been developed using segmented flow analysis. This approach enhanced 2-fold the throughput of the published direct injection and flow injection methods, improved sample utilization 3-fold, and was applicable to high-field NMR facilities with long transfer lines between the sample handler and NMR magnet. Sample volumes of 2 microL (10-30 mM, approximately 10 microg) were drawn from a 96-well microtiter plate by a sample handler, then pumped to a 0.5-microL microcoil NMR probe as a queue of closely spaced "plugs" separated by an immiscible fluorocarbon fluid. Individual sample plugs were detected by their NMR signal and automatically positioned for stopped-flow data acquisition. The sample in the NMR coil could be changed within 35 s by advancing the queue. The fluorocarbon liquid wetted the wall of the Teflon transfer line, preventing the DMSO samples from contacting the capillary wall and thus reducing sample losses to below 5% after passage through the 3-m transfer line. With a wash plug of solvent between samples, sample-to-sample carryover was <1%. Significantly, the samples did not disperse into the carrier liquid during loading or during acquisitions of several days for trace analysis. For automated high-throughput analysis using a 16-second acquisition time, spectra were recorded at a rate of 1.5 min/sample and total deuterated solvent consumption was <0.5 mL (1 US dollar) per 96-well plate.

Combinatorial Chemistry Techniques↗

PubMedDate not supplied

Off-line coupling of high-resolution capillary electrophoresis to MALDI-TOF and TOF/TOF MS.

High-resolution capillary electrophoresis has been coupled to MALDI-TOF and TOF/TOF MS through off-line vacuum deposition onto standard stainless steel MALDI targets. This off-line approach allowed the decoupling of the separation from the MS analysis, thus allowing each to be independently optimized in terms of time. Using BSA tryptic digest as a model sample, the deposited streaks, roughly 100-microm wide, were first analyzed in the MS mode, consuming only a fraction of the sample. After data analysis, segments of the deposited trace, containing unidentified peptides, as well as several species chosen for sequence confirmation, were reanalyzed in the MS/MS mode using MALDI-TOF/TOF MS. Additionally, it is shown that the shot-to-shot reproducibility of the vacuum-deposited trace (5% RSD) is 1 order of magnitude lower than that found for the standard dried droplet method. Moreover, a linear dependence of signal intensities (relative to an internal standard) over 3 orders of magnitude was found for a peptide sample with concentrations ranging from 1 to 1000 nM. This paper demonstrates the potential of off-line coupling of high-resolution separations to MALDI-MS and MALDI-MS/MS using vacuum deposition for the analysis of complex peptide mixtures from protein digests.

Electrophoresis, Capillary↗

PubMedDate not supplied

Proteomic analysis of Trypanosoma cruzi developmental stages using isotope-coded affinity tag reagents.

Comparative proteome analysis of developmental stages of the human pathogen Trypanosoma cruzi was carried out by isotope-coded affinity tag technology (ICAT) associated with liquid cromatography-mass spectrometry peptide sequencing (LC-MS/MS). Protein extracts of the protozoan trypomastigote and amastigote stages were labeled with heavy (D8) and light (D0) ICAT reagents and subjected to cation exchange and avidin affinity chromatographies followed by LC-MS/MS analysis. High confidence sequence information and expression levels for 41 T. cruzi polypeptides, including metabolic enzymes, paraflagellar rod components, tubulins, and heat-shock proteins were reported. Twenty-nine proteins displayed similar levels of expression in both forms of the parasite, nine proteins presented higher levels in trypomastigotes, whereas three were more expressed in amastigotes.

Amino Acid Sequence↗

PubMedDate not supplied

Proteomic analysis of ductal carcinoma of the breast using laser capture microdissection, LC-MS, and 16O/18O isotopic labeling.

The goal of this study was the development of a method for quantitative expression proteomics on the limited sample amounts obtained through laser capture microdissection (LCM) of tissues, e.g., approximately 10 000 cells, which typically contain roughly 1-4 microg protein. The 16O/18O labeling method was selected as an approach to measure differential expression. A sample preparation protocol including lysis, digestion and 16O/18O labeling was first developed for LCM cell samples. The selected protocol was examined using two LCM caps of 10 000 cells from invasive ductal carcinoma of the breast and shown to be repeatable. A further test of LC-IT-MS/MS in combination with the 16O/18O post-digestion labeling method for studying low level samples was conducted first on a single protein (BSA) and then on a 5-standard protein mixture digest of different protein amounts, each with a total content approximately 1 microg. Next, protein expression was compared between 10 000 cells, each of microdissected normal ductal epithelium and metastatic ductal carcinoma, using the developed method. The proteins from the microdissected cells were extracted, precipitated, digested with trypsin and then 16O/18O labeled. The normal and metastatic cell samples were analyzed using reversed phase LC-ESI-MS/MS on the ion trap mass spectrometer. A total of 76 proteins were identified. Some, such as mitochondrial isocitrate dehydrogenase, actin and 14-3-3 protein xi/delta were found to be significantly up-regulated in the breast tumor cells.

Amino Acid Sequence↗

PubMedDate not supplied

Proteome of Methanosarcina acetivorans Part II: comparison of protein levels in acetate- and methanol-grown cells.

Methanosarcina acetivorans is an archaeon isolated from marine sediments which utilizes a diversity of substrates for growth and methanogenesis. Part I of a two-part investigation has profiled proteins of this microorganism cultured with both methanol and acetate as growth substrates, utilizing two-dimensional gel electrophoresis and MALDI-TOF-TOF mass spectrometry. In this report, Part II, the analyses were extended to identify 34 proteins found to be present in different amounts between methanol- and acetate-grown M. acetivorans. Among these proteins are enzymes which function in pathways for methanogenesis from either acetate or methanol. Several of the 34 proteins were determined to have redundant functions based on annotations of the genomic sequence. Enzymes which function in ATP synthesis and steps common to both methanogenic pathways were elevated in acetate- versus methanol-grown cells, whereas enzymes that have a more general function in protein synthesis were in greater amounts in methanol- compared to acetate-grown cells. Several group I chaperonins were present in greater amounts in methanol- versus acetate-grown cells, whereas lower amounts of several stress related proteins were found in methanol- versus acetate-grown cells. The potential physiological basis for these novel patterns of protein synthesis are discussed.

Acetates↗

PubMedDate not supplied

Proteome of Methanosarcina acetivorans Part I: an expanded view of the biology of the cell.

Methanosarcina acetivorans is representative of the genus that is distinguished from all other methane-producing genera by extensive metabolic diversity predicted from the large genome. In Part I of this study, two-dimensional gel electrophoresis and MALDI-TOF-TOF mass spectrometry was used to investigate the proteome of methanol- or acetate-grown M. acetivorans, with the goal of an initial characterization of the diversity of the proteins synthesized. A total of 412 proteins were identified, representing nearly 10% of the ORFs, with nearly 30% conserved hypothetical or hypothetical. Of the 412 proteins, 188 were found in both acetate- and methanol-grown cells, 122 were detected only in acetate-grown cells, and 102 only in methanol-grown cells. The results revealed the expression of a remarkable number of redundant genes which encode enzymes involved in the pathways for methanogenesis from methanol or acetate, suggesting an important role for the unusually high percentage of redundant genes in Methanosarcina species. Evidence was obtained for synthesis of a sodium-transporting oxidoreductase in acetate-grown cells, with the potential to function in energy conservation. Several transcriptional regulatory proteins were identified that also function in the Bacteria domain, raising questions regarding their interaction with the Archaea/Eucarya-type basal transcription apparatus. In addition, a significant number of proteins involved in protein folding were shown to be synthesized in methanol- and acetate-grown cells. These studies provide the first examination of the protein diversity of M. acetivorans.

Acetates↗