Biomedical subjects

Jonathan W Cooper

Publications and source records attributed to Jonathan W Cooper.

7 recordsLinked to original sources

PubMed2006

Selective enrichment and ultrasensitive identification of trace peptides in proteome analysis using transient capillary isotachophoresis/zone electrophoresis coupled with nano-ESI-MS.

Besides the complexity in protein samples of biological origin, probably the greatest challenge presently facing comprehensive proteome analysis is related to the large variation of protein relative abundances (>6 orders of magnitude), having potential biological significance in mammalian systems. As demonstrated in this work, transient capillary ITP/zone electrophoresis (CITP/CZE) provides selective analyte enrichment through electrokinetic stacking and extremely high resolving power toward protein and peptide mixtures. The result of the CITP process is that major components may be diluted, but trace compounds are concentrated. The on-column transition of CITP to CZE minimizes additional band broadening while providing superior analyte resolution. Online coupling of transient CITP/CZE with nano-ESI-MS allows ultrasensitive detection of trace peptides at levels of subnanomolar concentration or subfemtomole mass in complex peptide mixtures. More importantly, selective enrichment of trace peptides enables the identification and sequence analysis of low-abundance peptides co-migrated with highly abundant species at a concentration ratio of 1:500,000. The combined CITP/CZE-nano-ESI-MS system is demonstrated to be at least one to two orders of magnitude more sensitive than that attained in conventional electrophoretic and chromatographic-based proteome technologies over a wide dynamic concentration range, potentially allowing comprehensive analysis of protein profiles within a small cell population and limited tissue samples using conventional mass spectrometers. Furthermore, the speed of CITP/CZE separation and the lack of column equilibration in CITP/CZE not only improve the throughput of proteome analysis, but also facilitate its seamless integration with other separation technologies in a multidimensional protein identification platform.

Cytochromes c↗

PubMed2004

Integrated and ultrasensitive gel protein identification.

An integrated gel protein identification technology is developed and demonstrated for the effective ( approximately 90% recovery), rapid (less than 5 min), and sensitive identification (as low as 1 ng gel protein loading) of gel-resolved proteins using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This integrated technology involves on-line combination of electronic protein transfer with nanoscale proteolytic digestion in a capillary platform, enabling electrokinetic-based protein extraction and stacking, real-time proteolytic cleavage of extracted proteins, and direct deposition of protein digests onto MALDI targets. By revisiting the yeast two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in similar isoelectric point and molecular mass ranges as studied by Gygi and co-workers (Gygi, S. P.; Corthals, G. L.; Zhang, Y.; Rochon, Y.; Aebersold, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9390-9395), we are additionally able to identify a large number of low abundance proteins with codon adaptation index (CAI) values of <0.2 and increase the proteome coverage to nearly 50%. The CAI value distribution for identified yeast proteins now more closely approximates that predicted for the entire yeast proteome. We further note that the current single-capillary methodology can be easily expanded to a multiplexed capillary platform as a ultrahigh throughput and greatly effective tool for linking 2-D PAGE with MS, particularly for the analysis of low-abundance proteins.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2004

Electronic gel protein transfer and identification using matrix-assisted laser desorption/ionization-mass spectrometry.

An electronic protein transfer technique is described for achieving the rapid and efficient recovery of sodium dodecyl sulfate (SDS)-protein complexes from polyacrylamide gels. This process involves the use of small-dimension capillaries in physical contact with a resolved protein band within the polyacrylamide gel, providing a large potential drop and high electric field strength at the capillary/gel interface. Several factors controlling the electronic protein transfer, including the applied electric field strength, the electrophoresis buffer concentration, and the capillary dimension, are studied to further enhance the use of field-amplification for sample stacking of extracted SDS-protein complexes. As a result of sample stacking, the extracted proteins from a 50 ng gel loading are present in a narrow ( approximately 80 nL) and highly concentrated (0.46 mg/mL or 3.3 x 10(-5) M for cytochrome c) solution plug. Three model proteins with molecular mass ranging from 14 kDa (cytochrome c) to 116 kDa (beta-galactosidase) are stained by Coomassie blue and electrophoretically extracted from gels with protein loadings as low as 50 ng. The capillary format of the electronic protein transfer technique allows direct deposition of extracted proteins onto a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) target. Various matrices and solvent compositions are evaluated for the analysis of extracted and concentrated SDS-protein complexes using MALDI-MS. The electronic protein transfer technique, when operated under optimized conditions, is demonstrated for the effective (>70% recovery), speedy (less than 5 min), and sensitive MS identification of gel resolved proteins (as low as 50 ng).

Cytochromes c↗

PubMed2004

Recent advances in capillary separations for proteomics.

The sequencing of several organisms' genomes, including the human's one, has opened the way for the so-called postgenomic era, which is now routinely coined as "proteomics". The most basic task in proteomics remains the detection and identification of proteins from a biological sample, and the most traditional way to achieve this goal consists of protein separations performed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Still, the 2-D PAGE-mass spectrometry (MS) approach remains lacking in proteome coverage (for proteins having extreme isoelectric points or molecular masses as well as for membrane proteins), dynamic range, sensitivity, and throughput. Consequently, considerable efforts have been devoted to the development of non-gel-based proteome separation technologies in an effort to alleviate the shortcomings in 2-D PAGE while reserving the ability to resolve complex protein and peptide mixtures prior to MS analysis. This review focuses on the most recent advances in capillary-based separation techniques, including capillary liquid chromatography, capillary electrophoresis, and capillary electrokinetic chromatography, and combinations of multiples of these mechanisms, along with the coupling of these techniques to MS. Developments in capillary separations capable of providing extremely high resolving power and selective analyte enrichment are particularly highlighted for their roles within the broader context of a state-of-the-art integrated proteome effort. Miniaturized and integrated multidimensional peptide/protein separations using microfluidics are further summarized for their potential applications in high-throughput protein profiling toward biomarker discovery and clinical diagnosis.

Chromatography, Liquid↗

PubMed2003

Membrane-based nanoscale proteolytic reactor enabling protein digestion, peptide separation, and protein identification using mass spectrometry.

A miniaturized trypsin membrane reactor housed inside a commonly used capillary fitting is developed and demonstrated for enabling rapid and sensitive protein identification by on-line proteolytic digestion and analysis of protein digests using nano-ESI-MS and MALDI-MS. The design and assembly of the capillary fitting-based trypsin membrane reactor are straightforward and highly robust, without the need for expensive fabrication technology and procedures. The resultant protein digests can also be further concentrated and resolved using capillary reversed-phase liquid chromatography or transient capillary isotachophoresis/zone electrophoresis prior to the mass spectrometric analysis in an integrated platform. By comparing these results with the results obtained from our previous studies using plastic microfluidics (Gao et al., Anal. Chem. 2001, 73, 2648-2655), significant reduction in dead volume and sample consumption can be achieved using this newly developed tryptic digestion station. This nanoscale reaction system enables rapid proteolytic digestion in seconds instead of hours for a protein concentration of less than 10(-8) M, consumes very little sample (< or = 5 fmol), and offers capillary interfaces with various separation and mass spectrometry techniques. The ultrafast enzymatic turnover for attaining complete peptide coverage in protein identification is contributed by the highly porous structure of the membrane media, providing excessive trypsin loading while eliminating the constraints of diffusion-limited reaction kinetics.

Indicators and Reagents↗

PubMed2002

Miniaturized membrane-based reversed-phase chromatography and enzyme reactor for protein digestion, peptide separation, and protein identification using electrospray ionization mass spectrometry.

A commonly used capillary fitting is employed for housing miniaturized membrane chromatography for performing reversed-phase peptide separations. By placing a hydrophobic and porous polyvinylidene fluoride membrane around the end of a polymer sleeve, the assembly of capillary fitting not only provides the stationary phase, but also establishes the necessary flow paths using capillary connections. The miniaturized membrane chromatography system is coupled with a micro-enzyme reactor containing immobilized trypsins for performing rapid protein digestion, peptide separation, and protein identification using electrospray ionization mass spectrometry. Separation performance of cytochrome c digest in miniaturized membrane chromatography is compared with the results obtained from micro-LC and capillary LC. The efficacy and the potentials of miniaturized membrane chromatography in tryptic mapping are reported. The use of miniaturized membrane chromatography allows significant reduction in sample consumption together with enhanced detection sensitivity. By minimizing the void volume in miniaturized membrane chromatography, the elution times of cytochrome c peptides are significantly shortened in this study in comparison with our previous results, and are comparable with those in micro-LC and capillary LC using considerably higher mobile phase flow-rates.

Animals↗

PubMed2002

Gel protein capillary extraction apparatus. electronic protein transfer.

A gel protein capillary extraction apparatus is developed and demonstrated for its rapid and effective transfer of SDS-protein complexes from polyacrylamide gel to a fused-silica capillary. The small dimensions of capillary columns permit the application of high voltages for achieving rapid and effective transfer of gel proteins. Furthermore, the fused-silica capillaries are internally coated with polyacrylamide for the elimination of electroosmotic pumping and protein adsorption onto the capillary wall. The extracted proteins are present in a highly concentrated solution plug as the result of field amplification and sample stacking during the extraction process. Three model proteins, including cytochrome c (14 kDa), ovalbumin (45 kDa), and beta-galactosidase (116 kDa), are visualized using coomassie blue staining and electrophoretically extracted from the gels with protein loading as low as 50 ng. The SDS-cytochrome c complexes extracted from a 50-ng protein loading are concentrated in a 30-nL solution plug inside the capillary with an estimated concentration of 0. 1 mg/mL or 10(-5) M. The capillary format allows the straightforward integration of a miniaturized trypsin-membrane reactor for on-line proteolytic digestion and ESI-MS analysis for protein/peptide identification.

Calibration↗