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D R Knapp

Publications and source records attributed to D R Knapp.

At least 19 recordsLinked to original sources
PubMed2001

Mass spectrometric analysis of integral membrane proteins at the subnanomolar level: application to recombinant photopigments.

Integral membrane proteins produced by eukaryotic expression systems are a subject of much current interest in biomedical investigation. Due to the low efficiency of their expression and the limited quantity of the expressed to the total amount of the membrane proteins, they have evaded mass spectrometric analysis. The methodology previously developed for mass spectrometric analysis of integral membrane proteins required proteins that were obtained relatively pure from their native membranes. The previously developed methodology has been modified and applied to the analysis of subnanomolar samples of rhodopsin. Bovine rhodopsin purified by affinity chromatography, from native membranes and from a eukaryotic expression system, was successfully analyzed, obtaining complete sequence coverage for the detection and localization of posttranslational modifications. The methodology presented here will enable mass spectrometric analysis of subnanomolar levels of photopigments or other integral membrane proteins either from their native membranes or as products of expression systems.

Amino Acid Sequence↗
PubMed2001

Microfabrication of polydimethylsiloxane electrospray ionization emitters.

Microfabricated polydimethylsiloxane (PDMS) emitters for electrospray ionization mass spectrometry (ESI-MS) were implemented as tips along the edge of the PDMS device by three methods which utilize soft lithography processes. These microfabrication methods for producing PDMS emitters as an integral part of a microfluidic device will facilitate development of more complex microfluidic analysis systems using ESI-MS.

Dimethylpolysiloxanes↗
PubMed2001

Miniaturized multichannel electrospray ionization emitters on poly(dimethylsiloxane) microfluidic devices.

A multichannel electrospray ionization (ESI) emitter was fabricated as part of a poly(dimethylsiloxane) (PDMS) microfluidic device using a three-layer photoresist process which also produces a self-alignment system to make a bonding between the top and bottom PDMS parts. The prototype device (2 cm high x 5 cm wide x 5 cm long) had 16-channels (30 microm wide x 50 microm deep) with emitters of 1 mm length and 60 degrees point angle. The PDMS emitter tips enabled interfacing the device to ESI-mass spectrometry; a stable electrospray from the tips was performed with limits of detection under 1 microM for reference peptides (adrenocorticotropic hormone fragment 1-17, angiotensin I and III).

Adrenocorticotropic Hormone↗
PubMed2001

Microfabricated PDMS multichannel emitter for electrospray ionization mass spectrometry.

A novel microfabricated multichannel emitter for electrospray ionization mass spectrometry (ESI-MS) was implemented with polydimethylsiloxane (PDMS) using a soft lithography technique. The emitters are formed as electrospray tips along a thin membrane on the edge of the device with channels of 100 microm x 30 microm dimensions. The electrospray performance of the PDMS emitters for a single channel device and a four channel device interfaced with a time-of-flight mass spectrometer was evaluated for detecting the molecular weight of reference peptides (angiotensin I and bradykinin). The emitters were durable at the flow rate of 1-20 microL min(-1) for more than 30 h of continuous electrospray with limit of detection of 1 microM (S/N 18). This microfabrication method for a PDMS multichannel emitter as an integral part of a microfluidic device will facilitate development of more complex microfluidic analysis systems using ESI-MS.

Journal Article↗
PubMed2000

Mass spectrometric analysis of rhodopsin from light damaged rats.

PURPOSE: It is well established that the retina is damaged by intense visible light. Rhodopsin has been proposed to be involved in this process. We therefore undertook to examine whether rhodopsin isolated from light damaged animals is structurally altered at the molecular level. METHODS: Dark reared and dim cyclic light reared 8 week old Sprague-Dawley rats were exposed to intense visible light and sacrificed immediately or 24 h after exposure together with unexposed control animals reared under the same conditions. Rod outer segments were isolated by sucrose gradient ultracentrifugation, their membranes treated with urea, then washed with Tris buffer. The rhodopsin preparations were then reduced, pyridylethylated, delipidated, and cleaved with CNBr. Reversed phase HPLC was used to separate the fragments, and the effluent was analyzed online with a Finnigan LCQ ion trap mass spectrometer. C-terminal phosphorylation was investigated following Asp-N cleavage. MALDI-TOF mass spectrometry was used for the identification of glycosylation. RESULTS: The rat rhodopsin protein was mapped with the exception of two single amino acid fragments. The reported sequence was confirmed with the exception of the controversial T/S320 residue, which was found to be a threonine. Mono-, di-, tri-, and tetraphosphorylated forms of rhodopsin were found in the light damaged animals. Three sites of phosphorylation were confirmed with MS/MS (tandem mass spectral) data. Single or double phosphorylations were found among these three sites, in various combinations. Dark adaptation completely reversed the phosphorylation in all light damaged animals. Other posttranslational modifications were as previously reported. CONCLUSIONS: Our results indicate that intense visible light exposure of rats does not lead to oxidative or other primary structural alterations in the rhodopsin protein of rod outer segments. We also report that the mutated rhodopsin (P23H) is present in rat rod outer segments from heterozygous animals and that residue 320 in both normal and mutated rhodopsins is threonine, not serine.

Amino Acid Sequence↗
PubMed2000

Intrahelical arrangement in the integral membrane protein rhodopsin investigated by site-specific chemical cleavage and mass spectrometry.

Site-specific cleavage on the interhelical loop I on the cytoplasmic face of rhodopsin has been observed after activation of a Cu-phenanthroline tethered cleavage reagent attached on the cytoplasmic loop IV. The characterization of the reaction products by mass spectrometry, both of the membrane-bound protein and of the CNBr-cleaved peptides, allows the site of cleavage to be determined precisely. The specific cleavage of the peptide bond between Q64 and H65 on loop I leaves the N-terminal peptide (M1-Q64) intact, confirmed by MALDI-MS detection of the two N-linked glycosyl groups near the N-terminus of rhodopsin. The limited extension of the tether side chain requires a interresidue distance between the cleavage site, Q64, and the site of ligand attachment, C316, of less than 12 A. Upon photoactivation of the receptor, no change in the cleavage pattern is observed; however, a simulated Meta II intermediate activation state indicates a much more complex cleavage pattern. The development of this cleavage method, previously used primarily as a "chemical nuclease", in combination with mass spectrometry, may provide a powerful method on membrane protein conformation studies that can be used to complement other biophysical characterizations.

Amino Acid Sequence↗
PubMed1998

A major G protein alpha O isoform in bovine brain is deamidated at Asn346 and Asn347, residues involved in receptor coupling.

The structural differences between two major forms of the alpha subunit of the heterotrimeric G protein GO were found to be due to deamidation of either of two Asn residues near the C-terminus of the proteins, in a region involved in receptor recognition. GO is the most abundant heterotrimeric G protein in mammalian brain. Two forms of the protein, GOA and GOB, are known to be generated by alternative splicing of a single GOalpha gene. A third isoform, alphaOC, represents about 1/3 of the alphaO protein in brain and is related to alphaOA, from which it is thought to be generated by protein modification. Mass spectrometry and chemical derivatization of tryptic fragments of the proteins were used to localize the structural difference between alphaOA and alphaOC to a C-terminal peptide. Sequence analysis of a C-terminal chymotryptic fragment both by ion trap mass spectrometry and by Edman degradation identified Asn346 and Asn347 of alphaOA as alternative deamidation sites in alphaOC. These structural differences have immediate implications for G protein function, as they occur in a conformationally sensitive part of the protein involved in receptor recognition and activation. Since Asn347 is a conserved residue present in most G protein alpha subunits outside the alphas family, these observations may have general significance for many G proteins. Deamidation may be a component of a novel process for modifying or adapting cellular responses mediated by G proteins.

Amides↗
PubMed1998

Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin.

Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G-protein coupled receptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)-MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins.

Amino Acid Sequence↗
PubMed1994

Observation of large multimers in the electrospray ionization mass spectrometry of peptides.

Large multimeric ions were detected from electrospray ionization (ESI) mass spectrometry of peptides, using a quadrupole mass spectrometer of standard mass range. Multimers, up to heptamers, were seen for angiotensin I; and multimers, up to pentamers, were observed for renin substrate. The appearance of the multimeric species could be controlled by manipulation of sampling conditions in the ESI interface. The relevance of the observation of large multimers to proposed mechanisms of ESI is discussed. Implications of the observation of large multimers to efforts to deduce higher order structure of biomolecules from ESI mass spectrometric experiments are also noted.

Amino Acid Sequence↗
PubMed1994

Azidotetrafluorophenyl retinal analogue: synthesis and bacteriorhodopsin pigment formation.

The retinal derivative, all-trans-9-(4-azido-2,3,5,6-tetrafluorophenyl)-3,7- dimethyl-2,4,6,8-nonatetraenal, was synthesized by two routes as a potential photoactivatable cross-linking agent for studies in bacteriorhodopsin (BR) of the chromophore interaction with its apoprotein. The retinal analogue formed a stable, moderately functional BR pigment confirming that the ring cavity of the retinal binding site has a significant tolerance for derivatization on that portion of the molecule. Attempts to cross-link the azido chromophore to the protein by photoactivation were unsuccessful. The electron delocalization effect of the conjugated polyene side chain of the retinal appears to interfere with the formation or reactivity of the nitrene intermediate to the extent that photoactivated cross-linking is not achieved. These results demonstrate a limitation to the use of fluorinated aryl azides as photoaffinity reagents.

Bacteriorhodopsins↗
PubMed1994

Evaluation of photoaffinity labeled sites of bacteriorhodopsin using molecular modeling.

Three successful photoaffinity labeling experiments of bacteriorhodopsin (bR) have been reported that used photoactivatible analogs of retinal to label the retinal binding site of the protein. Using molecular modeling techniques, the information about the retinal binding site derived from these studies is compared to the retinal binding site as defined by Henderson et al. (1990) using electron diffraction data. This comparison suggests some limitations to the use of photoaffinity labeling experiments for the determination of high resolution structural information.

Affinity Labels↗
PubMed1993

A site on transducin alpha-subunit of interaction with the polycationic region of cGMP phosphodiesterase inhibitory subunit.

Activation of cGMP phosphodiesterase (PDE) by the rod G-protein transducin is a key event in visual signal transduction in vertebrate photoreceptor cells. Interaction between the GTP-bound form of the alpha-subunit of transducin (alpha t*) and the PDE inhibitory gamma-subunit (P gamma) is a major component of PDE activation. The central polycationic region of P gamma, P gamma-24-45, has been implicated as one of the sites involved in alpha t*.P gamma interaction. Here we determine the site on alpha t* that interacts with P gamma-24-45 using a photo-cross-linking approach. The synthetic peptides Cys(ACM)Tyr-P gamma-24-45-Cys (where ACM indicates acetamidomethyl group) and Cys-P gamma-24-45 were labeled with 4-(N-maleimido)benzophenone at the COOH and NH2 termini, respectively, and then cross-linked to alpha t. When the photoprobe was attached to the COOH terminus of the peptide, a specific high yield cross-linked product (80%) was formed between the peptide and alpha t GTP gamma S (guanosine 5'-O-(thiotriphosphate)). A lower yield of cross-linking (35%) was seen between the peptide and alpha t GDP. The site of cross-linking between Cys(ACM)Tyr-P gamma-24-45-Cys and alpha t GTP gamma S was localized to within alpha t-306-310 using a variety of chemical and proteolytic cleavages of the cross-linked product, analysis of the fragments with SDS-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Mass spectrometric identification of phosphorylation sites in bleached bovine rhodopsin.

Deactivation of the visual cascade is initiated by the phosphorylation of rhodopsin. We report here identification of the two major sites of phosphorylation in bleached bovine rhodopsin using tandem mass spectrometry in conjunction with synthetic phosphopeptide standards. Both bleached and unbleached rod outer segments were cleaved with endoproteinase Asp-N to release the C-terminal fragment, residues 330-348, containing seven potential sites of phosphorylation. High-performance liquid chromatographic separation of soluble cleavage products from both unbleached and bleached rod outer segments gave a peak which was identified by tandem mass spectrometry and comparison to synthetic standards as monophosphorylated (serine 338) DDEASTTVSKTETSQVAPA. Present only in the chromatogram of bleached ROS were two peaks identified as monophosphorylated (serine 343) and diphosphorylated (serines 338 and 343) derivatives of DDEASTTVSKTETSQVAPA. These results identify serines 338 and 343 as the major sites of phosphorylation within the C-terminal region of bleached bovine rhodopsin and constitute the first example of mass spectrometric characterization of phosphorylation sites in a G-protein coupled receptor.

Amino Acid Sequence↗
PubMed1993

Mass spectrometric identification of modifications to human serum albumin treated with hydrogen peroxide.

Oxidized amino acid residues in human serum albumin exposed to hydrogen peroxide have been identified in tryptic peptides using liquid secondary ion mass spectrometry. Sites of oxidation identified include Cys34, Met123, Met298, Met446, and Met548. The extent of oxidation varied with location in the protein sequence, suggesting a relationship between oxidation and protein three-dimensional structure. The data presented here for human serum albumin demonstrate the utility of mass spectrometry in studying protein alterations. This type of information may be helpful in assessing the ability of proteins to act as antioxidants in biological systems which are subject to oxidant stress as in cases of inflammation and in the aging process.

Amino Acid Sequence↗
PubMed1993

Small-scale manual multiple peptide synthesis system. Application to phosphopeptide synthesis.

A system for small-scale (ca. 10-50 mumol) manual multiple peptide synthesis assembled from commercially available solid-phase extraction apparatus is described. This system was used to prepare (on a 15 mumol scale) the five monophosphorylated isomers of the peptide ASTTVSKTE, a proteolytic fragment of the C-terminal region of rhodopsin. The peptides were assembled using serine or threonine active esters without hydroxyl protection at the positions of phosphorylation. Phosphate groups were introduced using postassembly phosphitylation/oxidation according to a published procedure [Andrews, D.M., Kitchin, J. & Seale, P.W. (1991) Int. J. Peptide Protein Res. 38, 469-475; Staerkaer, G., Jakobsen, M.H., Olsen, C.E. & Holm, A., Tetrahedron Lett. 32, 5389-5392; Perich, J.W. (1992) Int. J. Peptide Protein Res. 40 134-140]. The reported system provides a relatively inexpensive approach to multiple peptide synthesis, including synthesis of phosphopeptides, for laboratories whose synthesis requirements do not justify investment in an automated multiple peptide synthesis instrument.

Amino Acid Sequence↗
PubMed1992

Palmitylation of a G-protein coupled receptor. Direct analysis by tandem mass spectrometry.

Bovine rhodopsin has been reported to be S-palmitylated at cysteines 322 and 323 (Ovchinnikov, Y. A., Abdulaev, N. G., and Bogachuk, A.S. (1988) FEBS Lett. 230, 1-5). Using a combination of enzymatic and chemical cleavage techniques in conjunction with tandem mass spectrometry, the sites of incorporation of the palmityl groups are shown. Bovine rhodopsin in disc membranes was digested with thermolysin to generate the C-terminal fragment (241-327), which was subsequently cleaved with cyanogen bromide to generate the peptide Val-Thr-Thr-Leu-Cys-Cys-Gly-Lys-Asn-Pro (318-327). A bis-S-palmitylated synthetic standard had the same retention time by reversed-phase high performance liquid chromatography as the isolated peptide and the same molecular weight (MH+1511.7) by liquid secondary ion mass spectrometry. Dithiothreitol reduction of both the isolated and the synthetic peptide cleaved the two thioester-linked palmityl groups to produce reduction products of the same appropriately decreased molecular weight (MH+1035.5). Tandem mass spectrometry of the isolated and the synthetic peptide identified the sites of attachment of the palmityl groups on cysteines 322 and 323. These results prove the modification of cysteines 322 and 323 with palmitic acid in bovine rhodopsin, and illustrate the utility of mass spectrometry to characterize the post-translational modifications in G-protein coupled receptors.

Amino Acid Sequence↗