Biomedical subjects

Mary F Lopez

Publications and source records attributed to Mary F Lopez.

4 recordsLinked to original sources

PubMed2003

Protein micro- and macroarrays: digitizing the proteome.

The early applications of microarrays and detection technologies have been centered on DNA-based applications. The application of array technologies to proteomics is now occurring at a rapid rate. Numerous researchers have begun to develop technologies for the creation of microarrays of protein-based screening tools. The stability of antibody molecules when bound to surfaces has made antibody arrays a starting point for proteomic microarray technology. To minimize disadvantages due to size and availability, some researchers have instead opted for antibody fragments, antibody mimics or phage display technology to create libraries for protein chips. Even further removed from antibodies are libraries of aptamers, which are single-stranded oligonucleotides that express high affinity for protein molecules. A variation on the theme of protein chips arrayed with antibody mimics or other protein capture ligand is that of affinity MS where the protein chips are directly placed in a mass spectrometer for detection. Other approaches include the creation of intact protein microarrays directly on glass slides or chips. Although many of the proteins may likely be denatured, successful screening has been demonstrated. The investigation of protein-protein interactions has formed the basis of a technique called yeast two-hybrid. In this method, yeast "bait" proteins can be probed with other yeast "prey" proteins fused to DNA binding domains. Although the current interpretation of protein arrays emphasizes microarray grids of proteins or ligands on glass slides or chips, 2-D gels are technically macroarrays of authentic proteins. In an innovative departure from the traditional concept of protein chips, some researchers are implementing microfluidic printing of arrayed chemistries on individual protein spots blotted onto membranes. Other researchers are using in-jet printing technology to create protein microarrays on chips. The rapid growth of proteomics and the active climate for new technology is driving a new generation of companies and academic efforts that are developing novel protein microarray techniques for the future.

Protein Array Analysis↗

PubMed2003

High-content proteomics: fluorescence multiplexing using an integrated, high-sensitivity, multiwavelength charge-coupled device imaging system.

The detection of proteins in 2-D gels and their subsequent identification by MS is still the "gold standard" in proteomics. Fluorescent detection has increasingly replaced colorimetric and radiometric detection on gels and blots. The reasons for this are multiple and varied and include higher sensitivity, better quantitation, increased dynamic range, speed, safety and ease of use. Unlike other methods, fluorescent protein detection is also typically very consistent in response from protein to protein and in many cases is compatible with MS methods for protein identification. The superior sensitivity and benefits achieved by fluorescent techniques have spurred the development of instrumentation capable of delivering precise, sensitive, high-resolution image acquisition over a wide variety of excitation and emission wavelengths. This report focuses on applications using the highly sensitive, charge-coupled device based ProXPRESS multilabel imager, readily configurable for image acquisition over a wide variety of wavelengths (380-700 nm and ultraviolet (UV)) using xenon lamp or UV excitation. The ability to simultaneously detect enzyme activities or protein modifications with different color fluorescent probes in addition to total protein amounts (multiplexing) allows the further mining of proteomic data content from a single set of protein samples. To this end, the development of instrumentation that enables a multiplexing strategy will become central to in-depth proteomic studies. The ProXPRESS maximizes the efficiency of experimental strategies that require flexibility and multicolor fluorescence detection.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2002

Applied proteomics: mitochondrial proteins and effect on function.

The identification of a majority of the polypeptides in mitochondria would be invaluable because they play crucial and diverse roles in many cellular processes and diseases. The endogenous production of reactive oxygen species (ROS) is a major limiter of life as illustrated by studies in which the transgenic overexpression in invertebrates of catalytic antioxidant enzymes results in increased lifespans. Mitochondria have received considerable attention as a principal source---and target---of ROS. Mitochondrial oxidative stress has been implicated in heart disease including myocardial preconditioning, ischemia/reperfusion, and other pathologies. In addition, oxidative stress in the mitochondria is associated with the pathogenesis of Alzheimer's disease, Parkinson's disease, prion diseases, and amyotrophic lateral sclerosis (ALS) as well as aging itself. The rapidly emerging field of proteomics can provide powerful strategies for the characterization of mitochondrial proteins. Current approaches to mitochondrial proteomics include the creation of detailed catalogues of the protein components in a single sample or the identification of differentially expressed proteins in diseased or physiologically altered samples versus a reference control. It is clear that for any proteomics approach prefractionation of complex protein mixtures is essential to facilitate the identification of low-abundance proteins because the dynamic range of protein abundance within cells has been estimated to be as high as 10(7). The opportunities for identification of proteins directly involved in diseases associated with or caused by mitochondrial dysfunction are compelling. Future efforts will focus on linking genomic array information to actual protein levels in mitochondria.

Animals↗

PubMed2002

An improved formulation of SYPRO Ruby protein gel stain: comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation.

SYPRO Ruby protein gel stain is compatible with a variety of imaging platforms since it absorbs maximally in the ultraviolet (280 nm) and visible (470 nm) regions of the spectrum. Dye localization is achieved by noncovalent, electrostatic and hydrophobic binding to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream proteomics techniques such as matrix-assisted laser desorption/ionisation-time of flight mass spectrometry is assured. The principal limitation of the original formulation of SYPRO Ruby protein gel stain, is that it was only compatible with a limited number of gel fixation procedures. Too aggressive a fixation protocol led to diminished signal intensity and poor detection sensitivity. This is particularly apparent when post-staining gels subjected to labeling with other fluorophores such as Schiff's base staining of glycoproteins with fluorescent hydrazides. Consequently, we have developed an improved formulation of SYPRO Ruby protein gel stain that is fully compatible with commonly implemented protein fixation procedures and is suitable for post-staining gels after detection of glycoproteins using the green fluorescent Pro-Q Emerald 300 glycoprotein stain or detection of beta-glucuronidase using the green fluorescent ELF 97 beta-D-glucuronide. The new stain formulation is brighter, making it easier to manually excise spots for peptide mass profiling. An additional benefit of the improved formulation is that it permits staining of proteins in isoelectric focusing gels, without the requirement for caustic acids.

Animals↗