23 research outputs found
Привлечение банков к венчурному финансированию в Республике Беларусь
Материалы XIII Междунар. науч.-техн. конф. студентов, магистрантов и молодых ученых, Гомель, 25–26 апр. 2013 г
Microfluidic electrocapture technology in protein and peptide analysis
After sequencing the genomes of several organisms, science in the
postgenomic era now aims at a thorough study of the proteins present in a
given tissue or organism. Since this task requires an enormous analytical
effort, integrated microfluidic systems are envisioned as the solution to
automated high throughput analysis of biomolecules.
This thesis is focused on a microfluidic methodology and device that
present several advantages over present technologies. The microfluidic
device utilizes an electric field to capture molecules traveling in a
flow stream. After capture, another medium is injected into the system
that is of a desirable chemical composition or carries reagents, which
are brought into contact with the captured molecules.
The microfluidic device was employed as a concentrator for capillary
electrophoresis (CE). Samples containing a mixture of proteins were
concentrated and injected into a CE instrument. Detection limits were
thereby improved from µM to nM protein levels.
The device was further applied to desalting and removal of contaminants
before MALDI-MS analysis. Polypeptides were captured followed by the
injection of a solvent suitable for NIS analysis. Significant desalting
and removal of CHAPS detergent was obtained for efficient analysis of
peptides and proteins by MALDI-MS.
In further study, the utilization of the electrocapture device to carry
out microreactions is described. After the capture of a target protein,
another medium containing enzymes and/or reagents is injected. Reduction,
alkylation, and trypsin digestion, as well as sample cleanup, were
carried out for peptide mass mapping by MALDI-MS.
The use of the electrocapture device as a separation tool is also
described. The separation process involves the capture and subsequent
sequential release of peptides according to their electrophoretic
mobility. Tryptic peptides from digestion of a mixture of proteins were
separated and analyzed by MALDI-MS.
A final study concerns the capture mechanism. It was found that
negatively charged molecules are in fact immobilized in the flow stream
due to a steady-state phenomenon created by the generation of areas with
different electric field strengths along the fluidic channel.
Herein we describe a flexible microfluidic device capable of processing
polypeptides to resolve key analytical problems in protein and peptide
analysis
Multistep microreactions with proteins using electrocapture technology
A method to perform multistep reactions by means of electroimmobilization of a target molecule in a microflow stream is presented. A target protein is captured by the opposing effects between the hydrodynamic and electric forces, after which another medium is injected into the system. The second medium carries enzymes or other reagents, which are brought into contact with the target protein and react. The immobilization is reversed by disconnecting the electric field, upon which products are collected at the outlet of the device for analysis. On-line reduction, alkylation, and trypsin digestion of proteins is demonstrated and was monitored by MALDI mass spectrometry
Biochemical Characterization of the Split Class II Ribonucleotide Reductase from Pseudomonas aeruginosa
The opportunistic pathogen Pseudomonas aeruginosa can grow under both aerobic and anaerobic conditions. Its flexibility with respect to oxygen load is reflected by the fact that its genome encodes all three existing classes of ribonucleotides reductase (RNR): the oxygen-dependent class I RNR, the oxygen-indifferent class II RNR, and the oxygen-sensitive class III RNR. The P. aeruginosa class II RNR is expressed as two separate polypeptides (NrdJa and NrdJb), a unique example of a split RNR enzyme in a free-living organism. A split class II RNR is also found in a few closely related gamma-Proteobacteria. We have characterized the P. aeruginosa class II RNR and show that both subunits are required for formation of a biologically functional enzyme that can sustain vitamin B12-dependent growth. Binding of the B12 coenzyme as well as substrate and allosteric effectors resides in the NrdJa subunit, whereas the NrdJb subunit mediates efficient reductive dithiol exchange during catalysis. A combination of activity assays and activity-independent methods like surface plasmon resonance and gas phase electrophoretic macromolecule analysis suggests that the enzymatically active form of the enzyme is a (NrdJa-NrdJb) 2 homodimer of heterodimers, and a combination of hydrogen-deuterium exchange experiments and molecular modeling suggests a plausible region in NrdJa that interacts with NrdJb. Our detailed characterization of the split NrdJ from P. aeruginosa provides insight into the biochemical function of a unique enzyme known to have central roles in biofilm formation and anaerobic growth
GEMMA analysis of protein oligomerization.
<p>Effectors induce protein dimerization of NrdJa. Without effector molecules, NrdJa is mostly in a monomeric state (81kDa, panel <b>A</b>). With dTTP (panel <b>B</b>) and dATP (panel <b>C</b>), the dimeric state dominates (173kDa).</p