PubMed Health⌕ Search

Biomedical subjects

Daniel R Knapp

Publications and source records attributed to Daniel R Knapp.

15 recordsLinked to original sources
PubMed2006

Palmitylation of cone opsins.

Palmitylation is a widespread modification in G-protein-coupled receptors and often a dynamic process. In rhodopsins, palmitylation is static on C322/C323. Red/green (M/LWS) cone opsins have no cysteines at corresponding positions and no palmitylation. Blue (SWS2) cone opsins have a single corresponding cysteine and mass spectrometric analysis showed partial palmitylation of salamander SWS2 cone opsin. Ultraviolet (SWS1) cone opsins have one corresponding cysteine, but only unpalmitylated opsin was observed for mouse and salamander. The results show that the static palmitylation found on rhodopsin is not found on cone opsins and suggest the possibility of an unidentified role for opsin palmitylation in cones.

Animals↗
PubMed2006

Plastic microchip liquid chromatography-matrix-assisted laser desorption/ionization mass spectrometry using monolithic columns.

A prototype array of monolithic liquid chromatography (LC) columns was prepared in a plastic microfluidic device for the off-line interface with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The microfluidic channels were fabricated on a cyclic olefin copolymer (COC) plate by hot embossing. An array of methacrylate monolithic columns was prepared in the microfluidic channels by UV-initiated polymerization. The deposition system employed a pulsed electric field to transfer the effluents from multiple columns directly onto MALDI targets with a non-contact deposition method reported by Ericson et al. [C. Ericson, Q.T. Phung, D.M. Horn, E.C. Peters, J.R. Fitchett, S.B. Ficarro, A.R. Salmon, L.M. Brill, A. Brock, Anal. Chem. 75 (2003) 2309]. To characterize the off-line interface of the multiple-channel microchip LC and the MALDI-MS for the analysis of peptide mixtures, the separation efficiency and reproducibility tests in each column were carried out by separating a peptide mixture from tryptic digested proteins and depositing the multiple effluents simultaneously on the MALDI target plate. Using a MALDI-TOF mass spectrometer with a mass accuracy of +/-1 Da for peptide assignments of digested bovine serum albumin (BSA), amino acid sequence coverage of around 59% was obtained for the microchip LC-MALDI-MS compared to 23% obtained by the MALDI-MS method without LC separation. In sensitivity tests for the detection of low abundance proteins in the presence of high concentration protein mixtures, as low as 10 fmol/mul (S/N = 10) of a spiked peptide in 1 microg of digested BSA could be detected. In the analysis of a mixture of three digested proteins (BSA, myoglobin, and cytochrome c), more than twice the amino acid sequence coverage was obtained for the microchip LC-MALDI-MS compared to MALDI-MS alone.

Cytochromes c↗
PubMed2006

Cell deposition system based on laser guidance.

We have designed a laser cell deposition system that employs the phenomenon of laser guidance to place single cells at specific points in a variety of in vitro environments. Here, we describe the components of the system: the laser optics, the deposition chamber, the microinjection cell feeding system and our custom system control software application. We discuss the requirements and challenges involved in laser guidance of cells and how our present system overcomes these challenges. We demonstrate that the patterning system is accurate within one micrometer by repeatedly depositing polymer microspheres and measuring their position. We demonstrate its ability to create highly defined living patterns of cells by creating a defined pattern of neurons with neurite extensions displaying normal function. We found that the positional accuracy of our system is smaller than the variations in cell size and pattern disruptions that occur from normal cell movement during substrate adhesion. The laser cell deposition system is a potentially useful tool that can be used to achieve site- and time-specific placement of an individual cell in a cell culture for the systematic investigation of cell-cell and cell-extracellular matrix interactions.

Animals↗
PubMed2006

Monolithic media in microfluidic devices for proteomics.

Considerable effort has been invested in the development of integrated microfluidic devices for fast and highly efficient proteomic studies. Among various fabrication techniques for the preparation of analytical components (separation columns, reactors, extractors, valves, etc.) in integrated microchips, in situ fabrication of monolithic media is receiving increasing attention. This is mainly due to the ease and simplicity of preparation of monolithic media and the availability of various precursors and chemistries. In addition, UV-initiated photopolymerization technique enables the incorporation of multiple analytical components into specified parts of a single microchip using photomasks. This review summarizes preparation methods for monolithic media and their application as microfluidic analytical components in microchips.

Microfluidic Analytical Techniques↗
PubMed2005

Proteomic analysis of bovine brain G protein gamma subunit processing heterogeneity.

We characterized the variable processing of the G protein gamma subunit isoforms associated with bovine brain G proteins, a primary mediator of cellular communication. Ggamma subunits were isolated from purified brain G proteins and characterized by Edman sequencing, by MALDI MS, by chemical and/or enzymatic fragmentation assayed by MALDI MS, and by MS/MS fragmentation and sequencing. Multiple forms of six different Ggamma isoforms were detected. Significant variation in processing was found at both the amino termini and particularly the carboxyl termini of the proteins. All Ggamma isoforms contain a carboxyl-terminal CAAX motif for prenylation, carboxyl-terminal proteolysis, and carboxymethylation. Characterization of these proteins indicates significant variability in the normal processing of all of these steps in the prenylation reaction, including a new variation of prenyl processing resulting from cysteinylation of the carboxyl terminus. These results have multiple implications for intracellular signaling mechanisms by G proteins, for the role of prenyl processing variation in cell signaling, and for the site of action and consequences of drugs that target the prenylation modification.

Animals↗
PubMed2005

Mass spectrometric analysis of integral membrane proteins at the subpicomolar level: application to rhodopsin.

Integral membrane proteins are among the most interesting molecules for biomedical research, as some of the most important cellular functions are inherently tied to biological membranes. One such example is the vast expanse of receptors on cell surfaces. However, due to difficulties in the biochemical purification and structure/function analysis of membrane proteins, caused by their hydrophobic or amphophilic nature, membrane proteins are still much less studied than soluble proteins. Our laboratory has successfully developed and applied a methodology for the mass spectrometric analysis of integral membrane proteins. Here, we present an improvement in the sensitivity of detection made possible by the advancement of mass spectrometric instrumentation and refinement of the chromatographic analysis. Subpicomolar samples of bovine rhodopsin purified from native membranes were successfully analyzed, obtaining complete sequence coverage and the detection and localization of posttranslational modifications. Therefore, it is demonstrated that the detection limits and sequence coverage for soluble and membrane proteins can be comparable. The methodology presented here allows mass spectrometric analysis of subpicomolar levels of photopigments or other integral membrane proteins either from their native membranes or as products of expression systems.

Animals↗
PubMed2004

Probing rhodopsin-transducin interactions by surface modification and mass spectrometry.

The interactions of rhodopsin and the alpha-subunit of transducin (G(t)) have been mapped using a surface modification "footprinting" approach in conjunction with mass spectrometric analysis employing a synthetic peptide corresponding to C-terminal residues 340-350 of the alpha-subunit of G(t), G(t)alpha(340-350). Membrane preparations of unactivated (Rh) and light-activated rhodopsin (Rh*), each in the presence or absence of G(t)alpha(340-350), were acetylated with the water-soluble reagent sulfosuccinimidyl acetate, and the extent of the acetylation was determined by mass spectrometry. By comparing the differences in acetylation among Rh, Rh*, and the Rh-G(t)alpha(340-350) and Rh*-G(t)alpha(340-350) complexes, we demonstrate that the surface exposure of the acetylation sites was reduced by the conformational change associated with light activation, and that binding of G(t)alpha(340-350) blocks acetylation sites on cytoplasmic loops 1, 2, and 4 of Rh*. In addition, we show evidence of interaction between the end of the C-terminal tail of rhodopsin and G(t)alpha in the unactivated state of rhodopsin.

Acetylation↗
PubMed2004

Capillary high-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic columns and carbon fiber electrospray ionization emitters.

Monolithic columns having long hydrocarbon chains were prepared by in-situ polymerization in capillary fused silica tubing. The capillary columns were coupled with a newly developed carbon fiber electrospray ionization (ESI) emitter for proteomic analysis using sheathless capillary HPLC-ESI mass spectrometry (MS). The sample loading capacity and chromatographic performance of the styrene-based monolithic column, which was prepared by photo-polymerization of octylstyrene (OS) and divinylbenzene (DVB) were compared with that of the methacrylate-based monolithic column composed of lauryl methacrylate (LMA) and ethylene dimethacrylate (EDMA). The sample loading ability of tryptic digested protein in poly-OS (POS)-DVB column was higher than that of poly-LMA (PLMA)-EDMA column, possibly due to the irregular and rugluous surface offering a greater surface area of POS-DVB stationary phase. The POS-DVB column also provided better separation efficiency in the separation of high concentration (10 microg) of tryptic digested albumin bovine serum (BSA). Due to the successful interface of a highly efficient monolithic column and a stable, durable carbon fiber emitter, low femtomole levels of peptides were successfully separated and identified in the presence of large amounts of tryptic digested protein.

Amino Acid Sequence↗
PubMed2004

Electrospray ionization with a pointed carbon fiber emitter.

A new type of electrospray ionization emitter employing a pointed carbon fiber has been developed for interfacing nanoliquid sampling techniques to mass spectrometry. The pointed carbon fiber protruding from an orifice with a surrounding hydrophobic surface confines a small Taylor cone at the tip, which generates a stable electrospray at the tip point. The small Taylor cone improves the electrospray efficiency thereby enhancing the detection limit. This emitter is rugged and able to generate stable electrospray over a wide range of flow rate, ESI voltage, and surface tension variation. Using a solution of angiotensin I, the carbon fiber emitter in 75-microm-i.d. fused-silica tubing was shown to give ion current comparable to that from a commercial 8 microm orifice nanospray emitter. Use of the emitter for ESI-MS/MS analysis of peptides was examined by infusing a mixture of cytochrome c and myoglobin tryptic digest peptides. Protein identification was demonstrated at the level of less than 1 fmol of the peptide consumed. The use of the carbon fiber emitter for interfacing monolithic capillary HPLC to MS was also demonstrated.

Animals↗
PubMed2002

11-cis-retinal reduces constitutive opsin phosphorylation and improves quantum catch in retinoid-deficient mouse rod photoreceptors.

Rpe65(-/-) mice produce minimal amounts of 11-cis-retinal, the ligand necessary for the formation of photosensitive visual pigments. Therefore, the apoprotein opsin in these animals has not been exposed to its normal ligand. The Rpe65(-/-) mice contain less than 0.1% of wild type levels of rhodopsin. Mass spectrometric analysis of opsin from Rpe65(-/-) mice revealed unusually high levels of phosphorylation in dark-adapted mice but no other structural alterations. Single flash and flicker electroretinograms (ERGs) from 1-month-old animals showed trace rod function but no cone response. B-wave kinetics of the single-flash ERG are comparable with those of dark-adapted wild type mice containing a full compliment of rhodopsin. Application (intraperitoneal injection) of 11-cis-retinal to Rpe65(-/-) mice increased the rod ERG signal, increased levels of rhodopsin, and decreased opsin phosphorylation. Therefore, exogenous 11-cis-retinal improves photoreceptor function by regenerating rhodopsin and removes constitutive opsin phosphorylation. Our results indicate that opsin, which has not been exposed to 11-cis-retinal, does not generate the activity generally associated with the bleached apoprotein.

Amino Acid Sequence↗
PubMed2002

Mass spectrometric analysis of porcine rhodopsin.

Rhodopsin is the dim light photosensitive pigment of animals. In this work, we undertook to study the structure of rhodopsin from swine and compare it with bovine and rat rhodopsin. Porcine rhodopsin was analyzed using methodology developed previously for mass spectrometric analysis of integral membrane proteins. Combining efficient protein cleavage and high performance liquid chromatography separation with the sensitivity of mass spectrometry (MS), this technique allows the observation of the full protein map and the posttranslational modifications of the protein in a single experiment. The rhodopsin protein from a single porcine eye was sequenced completely, with the exception of two single-amino acid fragments and one two-amino acid fragment, and the gene sequence reported previously was confirmed. The posttranslational modifications, similar to the ones reported previously for bovine and rat rhodopsin, were also identified. Although porcine rhodopsin has a high degree of homology to bovine and rat rhodopsins and most of their posttranslational modifications are identical, the glycosylation and phosphorylation patterns observed were different. These results show that rhodopsin from a single porcine eye can be characterized completely by MS. This technology opens the possibility of rhodopsin structural and functional studies aided by powerful mass spectrometric analysis, using the fellow eye as an internal control.

Amino Acid Sequence↗