Biomedical subjects

G J Opiteck

Publications and source records attributed to G J Opiteck.

7 recordsLinked to original sources

PubMed1998

Photochemical protease: site-specific photocleavage of hen egg lysozyme and bovine serum albumin.

Site-specific photocleavage of hen egg lysozyme and bovine serum albumin (BSA) by N-(l-phenylalanine)-4-(1-pyrene)butyramide (Py-Phe) is reported. Py-Phe binds to lysozyme and BSA with binding constants 2.2 +/- 0.3 x 10(5) M-1 and 6.5 +/- 0.4 x 10(7) M-1, respectively. Photocleavage of lysozyme and BSA was achieved with high specificity when a mixture of protein, Py-Phe, and an electron acceptor, cobalt(III) hexammine (CoHA), was irradiated at 344 nm. Quantum yields of photocleavage of lysozyme and BSA were 0.26 and 0.0021, respectively. No protein cleavage was observed in the absence of Py-Phe, CoHA, or light. N-terminal sequencing of the protein fragments indicated a single cleavage site of lysozyme between Trp-108 and Val-109, whereas the cleavage of BSA was found to be between Leu-346 and Arg-347. Laser flash photolysis studies of a mixture of protein, Py-Phe, and CoHA showed a strong transient with absorption centered at approximately 460 nm, corresponding to pyrene cation radical. Quenching of the singlet excited state of Py-Phe by CoHA followed by the reaction of the resulting pyrenyl cation radical with the protein backbone may be responsible for the protein cleavage. The high specificity of photocleavage may be valuable in targeting specific sites of proteins with small molecules.

Amino Acid Sequence↗

PubMed1998

Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping.

A two-dimensional liquid chromatographic system is described here which uses size-exclusion liquid chromatography (SEC) followed by reversed-phase liquid chromatography (RPLC) to separate the mixture of proteins resulting from the lysis of Escherichia coli cells and to isolate the proteins that they produce. The size-exclusion chromatography can be conducted under either denaturing or nondenaturing conditions. Peaks eluting from the first dimension are automatically subjected to reversed-phase chromatography to separate similarly sized proteins on the basis of their various hydrophobicities. The RPLC also serves to desalt the analytes so that they can be detected in the deep ultraviolet region at 215 nm regardless of the SEC mobile phase used. The two-dimensional (2D) chromatograms produced in this manner then strongly resemble the format of stained 2D gels, in that spots are displayed on a X-Y axis and intensity represents quantity of analyte. Following chromatographic separation, the analytes are deposited into six 96-well (576 total) polypropylene microtiter plates via a fraction collector. Interesting fractions are analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or electrospray mass spectrometry (ESI/MS) depending on sample concentration, which both yield accurate (2 to 0.02%) molecular weight information on intact proteins without any additional sample preparation, electroblotting, destaining, etc. The remaining 97% of a fraction can then be used for other analyses, such Edman sequencing, amino acid analysis, or proteolytic digestion and sequencing by tandem mass spectrometry. This 2D HPLC protein purification and identification system was used to isolate the src homology (SH2) domain of the nonreceptor tyrosine kinase pp60c-src and beta-lactamase, both inserted into E. coli, as well as a number of native proteins comprising a small portion of the E. coli proteome.

Bacterial Proteins↗

PubMed1997

Two-dimensional SEC/RPLC coupled to mass spectrometry for the analysis of peptides.

A two-dimensional liquid chromatography system is described here which uses size exclusion liquid chromatography (SEC) followed by reversed phase liquid chromatography (RPLC) to separate the mixture of peptides resulting from the enzymatic digestion of a protein. A novel LC/LC interface, using two RPLC columns in parallel rather than storage loops, joins the two chromatographic dimensions. This new interface design permits the use of conventional analytical diameter HPLC columns, 7.8 mm for SEC and 4.6 mm for RPLC, making construction and maintenance of this system very easy. The reversed phase chromatography utilizes 1.5 microns diameter, nonporous C-18 modified silica particles, which produce fast and efficient analyses. Following the high-resolution two-dimensional chromatographic separation, an electrospray mass spectrometer detects the peptide fragments. The mass spectrometer scans a 2000 m/z range to identify the analytes from their molecular weights. The analyses of tryptic digests of ovalbumin and serum albumin are each described.

Chromatography, Liquid↗

PubMed1997

Comprehensive on-line LC/LC/MS of proteins.

This is a description of a comprehensive two-dimensional liquid chromatography (LC) system for the separation of protein mixtures. This system uses cation-exchange chromatography followed by reversed-phase chromatography (RPLC). The two LC systems are coupled by an eight-port valve equipped with two storage loops and under computer control. The RPLC effluent is sampled by both a UV detector and an electrospray mass spectrometer. In this way, complex mixtures of large biomolecules can be rapidly separated, desalted, and analyzed for molecular weight in less than 2 h. The system's utility is demonstrated with a mixture of standards and an Escherichia coli cell lysate.

Chromatography, Ion Exchange↗

PubMed1997

Rapid separation and characterization of protein and peptide mixtures using 1.5 microns diameter non-porous silica in packed capillary liquid chromatography/mass spectrometry.

Octadecyl-modified 1.5 microns diameter non-porous silica particles were packed in 150 microns i.d. (360 microns o.d.) capillaries with lengths of 20 cm which were used to separate proteins and peptides generated from enzymatic digests of proteins. Gradients were produced using an exponential dilution method at pressures of 520 Bar (7500 psi) and electrospray ionization mass spectrometry was used for detection. This system was similar to packed capillary perfusion chromatography with respect to chromatographic resolution and analysis time and had a limit of detection comparable to traditional packed capillaries which use 5 microns diameter porous particles. The analyses required as little as 250 femtomol of protein or 500 femtomol of peptide on-column in approximately 30 min. This technique was then applied to verify the existence of an overexpressed protein in an E. coli cell lysate and to confirm the presence of four glycoforms of a peptide generated in the proteolytic digest of an antibody.

Chromatography, Ion Exchange↗

PubMed1997

A multidimensional approach to protein characterization.

When mass spectrometry (MS) is used to study protein primary structure, it is used in a "static" mode. That is, the information is derived from a single MS or MS-MS spectrum. Information about more complex protein structure or protein interactions can also be gained via MS. If a series of mass spectra is collected as something else in the experiment is changing, we increase the "dimensionality" of the MS data. For example, measuring mass spectra as a function of time after exposure of a protein to deuterated solvents can provide information about protein structure. Likewise, by measuring mass spectra of a protein as the concentration of a binding ligand is changed, one can infer the stoichiometry of the complex. Another important, but fundamentally different way of increasing the dimensionality of mass spectral data is by coupling the mass spectrometer to a one- or two-dimensional separation technique.

Mass Spectrometry↗

PubMed1994

Two-dimensional gel electrophoresis/liquid chromatography for the micropreparative isolation of proteins.

Although comprehensive, column-based two-dimensional separation techniques offer enormous resolving power for a complex mixture, they often lack the ability to isolate the separated species for further analysis (e.g., proteins for Edman sequencing). This paper describes a micropreparative two-dimensional separation system for the isolation of proteins from complex mixtures, such as cell lysates. The system is composed of commercially available equipment: continuous-elution tube gel electrophoresis as the first dimension followed by gradient elution, reversed-phase perfusion liquid chromatography as the second dimension with a two-loop sampling valve as the interface between dimensions. The two-dimensional electrophoresis/liquid chromatography system (2D-EP/LC) shows high resolution of proteins since each dimension has orthogonal separation mechanisms (electrophoresis, size/charge; LC, hydrophobicity). Identification of proteins for further analysis is accomplished by superimposing a grid on the computer-generated 3D image.

Animals↗