Matching Items (34)

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Peptide Sequencing Directly on Solid Surfaces Using MALDI Mass Spectrometry

Description

There are an increasing variety of applications in which peptides are both synthesized and used attached to solid surfaces. This has created a need for high throughput sequence analysis directly

There are an increasing variety of applications in which peptides are both synthesized and used attached to solid surfaces. This has created a need for high throughput sequence analysis directly on surfaces. However, common sequencing approaches that can be adapted to surface bound peptides lack the throughput often needed in library-based applications. Here we describe a simple approach for sequence analysis directly on solid surfaces that is both high speed and high throughput, utilizing equipment available in most protein analysis facilities. In this approach, surface bound peptides, selectively labeled at their N-termini with a positive charge-bearing group, are subjected to controlled degradation in ammonia gas, resulting in a set of fragments differing by a single amino acid that remain spatially confined on the surface they were bound to. These fragments can then be analyzed by MALDI mass spectrometry, and the peptide sequences read directly from the resulting spectra.

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Date Created
  • 2017-12-19

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Scalable high-density peptide arrays for comprehensive health monitoring

Description

There is an increasing awareness that health care must move from post-symptomatic treatment to presymptomatic intervention. An ideal system would allow regular inexpensive monitoring of health status using circulating antibodies

There is an increasing awareness that health care must move from post-symptomatic treatment to presymptomatic intervention. An ideal system would allow regular inexpensive monitoring of health status using circulating antibodies to report on health fluctuations. Recently, we demonstrated that peptide microarrays can do this through antibody signatures (immunosignatures). Unfortunately, printed microarrays are not scalable. Here we demonstrate a platform based on fabricating microarrays (~10 M peptides per slide, 330,000 peptides per assay) on silicon wafers using equipment common to semiconductor manufacturing. The potential of these microarrays for comprehensive health monitoring is verified through the simultaneous detection and classification of six different infectious diseases and six different cancers. Besides diagnostics, these high-density peptide chips have numerous other applications both in health care and elsewhere.

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Date Created
  • 2014-09-03

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Exploring the sequence space of a DNA aptamer using microarrays

Description

The relationship between sequence and binding properties of an aptamer for immunoglobulin E (IgE) was investigated using custom DNA microarrays. Single, double and some triple mutations of the aptamer sequence

The relationship between sequence and binding properties of an aptamer for immunoglobulin E (IgE) was investigated using custom DNA microarrays. Single, double and some triple mutations of the aptamer sequence were created to evaluate the importance of specific base composition on aptamer binding. The majority of the positions in the aptamer sequence were found to be immutable, with changes at these positions resulting in more than a 100-fold decrease in binding affinity. Improvements in binding were observed by altering the stem region of the aptamer, suggesting that it plays a significant role in binding. Results obtained for the various mutations were used to estimate the information content and the probability of finding a functional aptamer sequence by selection from a random library. For the IgE-binding aptamer, this probability is on the order of 10 [superscript − 10] to 10 [superscript − 9]. Results obtained for the double and triple mutations also show that there are no compensatory mutations within the space defined by those mutations. Apparently, at least for this particular aptamer, the functional sequence space can be represented as a rugged landscape with sharp peaks defined by highly constrained base compositions. This makes the rational optimization of aptamer sequences using step-wise mutagenesis approaches very challenging.

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Created

Date Created
  • 2007-12-01

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Peptide-Modified Surfaces for Enzyme Immobilization

Description

Background
Chemistry and particularly enzymology at surfaces is a topic of rapidly growing interest, both in terms of its role in biological systems and its application in biocatalysis. Existing protein

Background
Chemistry and particularly enzymology at surfaces is a topic of rapidly growing interest, both in terms of its role in biological systems and its application in biocatalysis. Existing protein immobilization approaches, including noncovalent or covalent attachments to solid supports, have difficulties in controlling protein orientation, reducing nonspecific absorption and preventing protein denaturation. New strategies for enzyme immobilization are needed that allow the precise control over orientation and position and thereby provide optimized activity.
Methodology/Principal Findings
A method is presented for utilizing peptide ligands to immobilize enzymes on surfaces with improved enzyme activity and stability. The appropriate peptide ligands have been rapidly selected from high-density arrays and when desirable, the peptide sequences were further optimized by single-point variant screening to enhance both the affinity and activity of the bound enzyme. For proof of concept, the peptides that bound to β-galactosidase and optimized its activity were covalently attached to surfaces for the purpose of capturing target enzymes. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activity and stability, as well as controlled protein orientation.
Conclusions/Significance
A simple method for immobilizing enzymes through specific interactions with peptides anchored on surfaces has been developed. This approach will be applicable to the immobilization of a wide variety of enzymes on surfaces with optimized orientation, location and performance, and provides a potential mechanism for the patterned self-assembly of multiple enzymes on surfaces.

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Created

Date Created
  • 2011-04-08

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Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

Description

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

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Created

Date Created
  • 2016-02-10

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TARGETING ADIPOSE TISSUE INFLAMMATION IN THE TREATMENT OF TYPE II DIABETES

Description

Diabesity is a global epidemic affecting millions worldwide. Diabesity is the term given to the link between obesity and Type II diabetes. It is estimated that ~90% of patients diagnosed

Diabesity is a global epidemic affecting millions worldwide. Diabesity is the term given to the link between obesity and Type II diabetes. It is estimated that ~90% of patients diagnosed with Type II diabetes are overweight or have struggled with excess body fat in the past. Type II diabetes is characterized by insulin resistance which is an impaired response of the body to insulin that leads to high blood glucose levels. Adipose tissue, previously thought of as an inert tissue, is now recognized as a major endocrine organ with an important role in the body's immune response and the development of chronic inflammation. It is speculated that adipose tissue inflammation is a major contributor to insulin resistance particular to Type II diabetes. This literature review explores the popular therapeutic targets and marketed drugs for the treatment of Type II diabetes and their role in decreasing adipose tissue inflammation. rAGE is currently in pre-clinical studies as a possible target to combat adipose tissue inflammation due to its relation to insulin resistance. Metformin and Pioglitazone are two drugs already being marketed that use unique chemical pathways to increase the production of insulin and/or decrease blood glucose levels. Sulfonylureas is one of the first FDA approved drugs used in the treatment of Type II diabetes, however, it has been discredited due to its life-threatening side effects. Bariatric surgery is a form of invasive surgery to rid the body of excess fat and has shown to normalize blood glucose levels. These treatments are all secondary to lifestyle changes, such as diet and exercise which can help halt the progression of Type II diabetes patients.

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Date Created
  • 2019-05

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Quinone Removal and Replacement within the Reaction Center Protein of Rhodobacter sphaeroides

Description

With a quantum efficiency of nearly 100%, the electron transfer process that occurs within the reaction center protein of the photosynthetic bacteria Rhodobacter (Rh.) sphaeroides is a paragon for understanding

With a quantum efficiency of nearly 100%, the electron transfer process that occurs within the reaction center protein of the photosynthetic bacteria Rhodobacter (Rh.) sphaeroides is a paragon for understanding the complexities, intricacies, and overall systemization of energy conversion and storage in natural systems. To better understand the way in which photons of light are captured, converted into chemically useful forms, and stored for biological use, an investigation into the reaction center protein, specifically into its cascade of cofactors, was undertaken. The purpose of this experimentation was to advance our knowledge and understanding of how differing protein environments and variant cofactors affect the spectroscopic aspects of and electron transfer kinetics within the reaction of Rh. sphaeroides. The native quinone, ubiquinone, was extracted from its pocket within the reaction center protein and replaced by non-native quinones having different reduction/oxidation potentials. It was determined that, of the two non-native quinones tested—1,2-naphthaquinone and 9,10- anthraquinone—the substitution of the anthraquinone (lower redox potential) resulted in an increased rate of recombination from the P+QA- charge-separated state, while the substitution of the napthaquinone (higher redox potential) resulted in a decreased rate of recombination.

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Date Created
  • 2015-12

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VDAP[a]: An R Package for Data Visualization and Motif Analysis on Peptide Microarrays

Description

Advances in peptide microarray technology have allowed for the creation of fast-paced and modular experiments within affinity ligand discovery. Previously, low density peptide arrays of 10,000 peptides were used to

Advances in peptide microarray technology have allowed for the creation of fast-paced and modular experiments within affinity ligand discovery. Previously, low density peptide arrays of 10,000 peptides were used to identify low affinity peptide ligands for a target protein; an approach that can be subsequently improved upon with a number of techniques. VDAP[a] offers more information about the relative affinity of protein-peptide interactions via signal intensity in contrast to high throughput screening (HTS) and display technologies which offer binary data. Now, high density peptide arrays with 130,000 to 330,000 peptides are available that allow screening across peptide libraries of greater diversity. With this increase in scale and diversity, faster analytical tools are needed to adequately characterize array data. Using the statistical power available in the R programming language, we have created a flexible analysis package that efficiently processes high density peptide array data from a variety of layouts, rank existing peptide hits, and utilize signal intensity data to generate new hits. This analysis provides a user-friendly method to efficiently analyze high density peptide array data, generate peptide leads for targeted therapeutic development, and further improve peptide array technologies.

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Created

Date Created
  • 2015-12

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Biophysical differentiation of MRSA and MSSA using Dielectrophoresis

Description

Dielectrophoresis has been shown in the recent past to successfully separate bioparticles of very subtle differences at high resolutions using biophysical forces. In this study, we test the biophysical differences

Dielectrophoresis has been shown in the recent past to successfully separate bioparticles of very subtle differences at high resolutions using biophysical forces. In this study, we test the biophysical differences of methicillin resistant and susceptible Staph. aureus that are known to have very similar genomes by using a modified gradient insulator-based dielectrophoresis device (g-iDEP). MRSA is commonly seen in hospitals and is the leading killer of infectious bacteria, claiming the lives of around 10,000 people annually. G-iDEP improves many capabilities within the DEP field including sample size, cost, ease of use and analysis time. This is a promising foundation to creating a more clinically optimized diagnostic tool for both separation and detection of bacteria in the healthcare field. The capture on-set potential for fluorescently tagged MRSA (801 ± 34V) is higher than fluorescently tagged MSSA (610 ± 32V), resulting in a higher electrokinetic to dielectrophoretic mobility ratio for MRSA. Since the strains have proven to be genomically similar through sequencing, it is reasonable to attribute this significant biophysical difference to the added PBP2a enzyme in MRSA. These results are consistent with other bacterial studied within in this device and have proven to be reproducible.

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Date Created
  • 2020-05

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Reduction of Carbon Dioxide with Cobalt and Iron Porphyrins

Description

The free-base tetra-tolyl-porphyrin and the corresponding cobalt and iron porphyrin complexes were synthesized and characterized to show that this class of compound can be promising, tunable catalysts for carbon dioxide

The free-base tetra-tolyl-porphyrin and the corresponding cobalt and iron porphyrin complexes were synthesized and characterized to show that this class of compound can be promising, tunable catalysts for carbon dioxide reduction. During cyclic voltammetry experiments, the iron porphyrin showed an on-set of ‘catalytic current’ at an earlier potential than the cobalt porphyrin’s in organic solutions gassed with carbon dioxide. The cobalt porphyrin yielded larger catalytic currents, but at the same potential as the electrode. This difference, along with the significant changes in the porphyrin’s electronic, optical and redox properties, showed that its capabilities for carbon dioxide reduction can be controlled by metal ions, allotting it unique opportunities for applications in solar fuels catalysis and photochemical reactions.

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Created

Date Created
  • 2016-05