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In JoVE (1)
Other Publications (17)
- Journal of the American Society for Mass Spectrometry
- Journal of the American Society for Mass Spectrometry
- The Journal of Organic Chemistry
- Journal of Combinatorial Chemistry
- Carbohydrate Research
- Journal of Chromatography. A
- Journal of the American Society for Mass Spectrometry
- Acta Crystallographica. Section C, Crystal Structure Communications
- Archives of Biochemistry and Biophysics
- The European Journal of Neuroscience
- Journal of Drug Targeting
- Protein Expression and Purification
- Journal of the American Society for Mass Spectrometry
- Protein Expression and Purification
- Biochemical and Biophysical Research Communications
- Biomacromolecules
- Gene
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Articles by Andreas Franz in JoVE
JoVE General
Microdissecção de Black Widow Aranha Silk glândulas produtoras de
Department of Biological Sciences, University of the Pacific
Aqui nós descrevemos uma estratégia eficiente para remover as glândulas produtoras de seda do abdômen da fêmea aranha viúva negra. Este procedimento permite o isolamento rápido da produção de seda sete diferentes glândulas de uma forma altamente purificada, um processo importante para os investigadores a estudar a produção de seda de aranha e montagem de fibra.
Other articles by Andreas Franz on PubMed
Evidence for Long-range Glycosyl Transfer Reactions in the Gas Phase
A long-range glycosyl transfer reaction was observed in the collision-induced dissociation Fourier transform (CID FT) mass spectra of benzylamine-labeled and 9-aminofluorene-labeled lacto-N-fucopentaose I (LNFP I) and lacto-N-difucohexaose I (LNDFH I). The transfer reaction was observed for the protonated molecules but not for the sodiated molecules. The long-range glycosyl transfer reaction involved preferentially one of the two L-fucose units in labeled LNDFH I. CID experiments with labeled LNFP I and labeled LNFP II determined the fucose with the greatest propensity for migration. Further experiments were performed to determine the final destination of the migrating fucose. Molecular modeling supported the experiments and reaction mechanisms are proposed.
Non-covalent Calixarene-amino Acid Complexes Formed by MALDI-MS
Non-covalent inclusion complexes formed between amino acids and derivatized calix[6]arenes are observed in MALDI mass spectrometry. The methyl, ethyl, and propyl ester derivatives of calix[6]arene yielded amino acid complexes, while the smaller calix[4]arene analogs did not. Similarly the underivatized calix[6]arene and calix[4]arene did not produce complexes. Amino acid complexes were observed for nearly all 20 amino acids in time-of-flight (TOF) analysis. In Fourier transform mass spectrometry (FTMS) analysis, however, only the most basic amino acids arginine, histidine, and lysine formed stable adducts. The complexes were abundant under matrix-assisted laser desorption ionization (MALDI) conditions, which suggested favorable interactions between host and guest.
Mild Synthesis of Disaccharidic 2,3-enopyranosyl Cyanides and 2-C-2-deoxy Pyranosyl Cyanides with Hg(CN)(2)/HgBr(2)/TMSCN
Lewis acid-catalyzed dimerization of mono- and disaccharidic per-O-acetylated glycals gave di- and tetrasaccharidic O-acetylated C-glycosides, respectively. 2,3-Enopyranosyl cyanides were obtained from per-O-acetylated glycals by a new, mild anomeric S(N)'-acetoxy displacement with Hg(CN)(2)/HgBr(2)/TMSCN. Per-O-acetylated 2-C-2-deoxy-pyranoses were converted into pyranosyl cyanides by the same reagent. An unprecedented acetic acid elimination from dimers with D-galacto- and L-fuco-configurations accompanied the S(N)-displacement under those conditions. A new set of (1)H NMR coupling constants for 2,3-enopyranosyl systems was used for configurational assignment of complicated tetrasaccharide mimics.
High-throughput One-bead-one-compound Approach to Peptide-encoded Combinatorial Libraries: MALDI-MS Analysis of Single TentaGel Beads
The identification of pharmacologically promising compounds (lead compounds) from combinatorial libraries is frequently limited by the throughput of the analytical technique employed. Fourier transform mass spectrometry (FTMS) offers high sensitivity, mass accuracy (m/Deltam > 500 000), and sequencing capabilities. A rapid and efficient method for high-throughput analysis of single beads from peptide-encoded combinatorial libraries with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is presented. Encoding peptides on single beads are identified and structurally characterized by MALDI time-of-flight (TOF) and ultrahigh-resolution MALDI Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. A strategy of on-probe sample preparation is developed to minimize handling of the beads.
C-H-deprotonation Mediated by a Remote Syn-axial Acetoxy Group--an Unprecedented Double Bond Formation Upon Cyanation of the Dimer from L-fucal
Replacement of the anomeric acetate by a cyanide group in the dimer of di-O-acetyl-L-fucal by the action of mild Lewis acid [Hg(CN)(2)-HgBr(2)-Me(3)SiCN], resulted not only in the desired transformation but also in the introduction of an additional double bond between C-2A and C-3A. Due to its configuration, the remote C-4A acetoxy group may facilitate the deprotonation by functioning as an internal base. 1H NMR spectroscopy and X-ray crystallography indicate that the conformations of both rings A and B and their relative orientation in the resulting C-linked disaccharidic glycosyl cyanide, 4-O-acetyl-2-C-(4-O-acetyl-2,3-dideoxy-alpha-L-threo-hex-2-enopyranosyl)-2,3-dideoxy-2-eno-alpha-L-fucopyranosyl cyanide, in solution are virtually identical to the crystal structure.
Improved Capillary Electrophoretic Separation and Mass Spectrometric Detection of Oligosaccharides
We have developed a CE method for the separation of structural isomers of oligosaccharides labeled with N-quaternized benzylamine. Oligosaccharides with reducing ends were derivatized with benzylamine by reductive amination followed by quaternization to yield a fixed cation label. The benzylamine-derivatized oligosaccharides were analyzed by CE-UV in ammonium acetate buffer and off-line matrix-assisted laser desorption ionization (MALDI) MS. The method was applied to a 1 nmol sample of a model oligosaccharide (LNDFH 1). From this sample a 38 fmol diluted standard was detected. The quaternization of benzylamine-labeled products significantly improved CE separation of neutral oligosaccharides along with several structural isomers. Two hexasaccharide isomers (LNDFH I and LNDFH II) were baseline resolved using an ammonium acetate buffer. This method was also applied successfully to the profiling of oligosaccharides released from the glycoprotein RNase B. The release of 6 pmol of glycans followed by workup showed the detection of all components, with one component corresponding to 100 fmol. Micropreparative collection of CE enabled successful off-line CE-MALDI-MS without additional sample clean up. This report provides a simple and rapid method to separate and analyze oligosaccharides.
MALDI-TOF and ESI-MS Analysis of Oligosaccharides Labeled with a New Multifunctional Oligosaccharide Tag
A new multifunctional oligosaccharide label with a 1 degree amino-group was synthesized and characterized. The oligosaccharide label was introduced into several neutral oligosaccharides by reductive amination, and the derivatives were analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and by electrospray ionization (ESI) mass spectrometry. It was demonstrated that the labeling reaction was satisfactory, and that as little as 50 pmol of starting material could be efficiently labeled with minimal loss to side reactions. A mixture of high-mannose N-glycans released from ribonuclease B was labeled. The label did not appear to interfere with structural characterization of the oligosaccharides by mass spectrometry. N-quaternization of the labeled oligosaccharides resulted in significantly increased sensitivity of detection with as little as 100 fmol on the probe detected. Deuterium coding of labeled oligosaccharide mixtures and relative abundance of mixture components was investigated. A protocol for the chromatographic separation of mixtures of labeled oligosaccharides by HPLC was developed and is reported here.
Tris(1,9-diethyladeninium) Triiodide Diiodide
X-ray analysis of the title compound reveals three crystallographically distinct cations of 1,9-diethyladeninium, two iodide anions and one triiodide anion in the asymmetric unit, giving six residues and the formula 3C9H14N5+.I3-.2I-. Standard purine nomenclature is used to identify the atoms of each adenine moiety. Hydrogen bonding is observed between atoms N6 and N7 of a pair of cations [N...N=2.885 (4)/2.902 (3) and 2.854 (3)/2.854 (3) A], with additional hydrogen bonding to I- anions via the other N6 H atom [N...I=3.708 (3), 3.738 (3) and 3.638 (3) A]. The triiodide anion is not involved in hydrogen bonding. The bond lengths and angles of the 1,9-diethyladeninium cations are compared with literature values and confirm the formation of the imine tautomer.
The Disulfide Bond Pattern of Salmon Egg Lectin 24K from the Chinook Salmon Oncorhynchus Tshawytscha
The disulfide bonds in the galactose-specific lectin SEL 24K from the egg of the Chinook salmon Oncorhynchus tshawytscha were determined by mass spectrometry. Four predictive in silico tools were used to determine the oxidation state of cysteines in the sequence and possible location of the disulfide bonds. A combination of tryptic digestion, HPLC separation, and chemical modifications were used to establish the location of seven disulfide bonds and one pair of free cysteines. After proteolysis, peptides containing one or two disulfide bonds were identified by reduction and mass spectral comparison. MALDI mass spectrometry was supported by chemical modification (iodoacetamide) and in silico digestion. The assignments of disulfide bonds were further confirmed by mass spectral fragmentation studies including in-source dissociation (ISD) and collision-induced dissociation (CID). The experimentally determined disulfide bonds and free Cys residues were only partially consistent with those generated by several automated public-domain algorithms.
Deglycosylated Anti-amyloid Beta Antibodies Reduce Microglial Phagocytosis and Cytokine Production While Retaining the Capacity to Induce Amyloid Beta Sequestration
Accumulation of amyloid beta (Abeta) is a pathological hallmark of Alzheimer's disease, and lowering Abeta is a promising therapeutic approach. Intact anti-Abeta antibodies reduce brain Abeta through two pathways: enhanced microglial phagocytosis and Abeta transfer from the brain to the periphery (Abeta sequestration). While activation of microglia, which is essential for microglial phagocytosis, is necessarily accompanied by undesired neuroinflammatory events, the capacity for sequestration does not seem to be linked to such effects. We and other groups have found that simple Abeta binding agents are sufficient to reduce brain Abeta through the sequestration pathway. In this study, we aimed to eliminate potentially deleterious immune activation from antibodies without affecting the ability to induce sequestration. The glycan portion of immunoglobulin is critically involved in interactions with immune effectors including the Fc receptor and complement c1q; deglycosylation eliminates these interactions, while antigen (Abeta)-binding affinity is maintained. In this study, we investigated whether deglycosylated anti-Abeta antibodies reduce microglial phagocytosis and neuroinflammation without altering the capacity to induce Abeta sequestration. Deglycosylated antibodies maintained Abeta binding affinity. Deglycosylated antibodies did not enhance Abeta phagocytosis or cytokine release in primary cultured microglia, whereas intact antibodies did so significantly. Intravenous injection of deglycosylated antibodies elevated plasma Abeta levels and induced Abeta sequestration to a similar or greater degree compared with intact antibodies in an Alzheimer's transgenic mouse model without or with Abeta plaque pathology. We conclude that deglycosylated antibodies effectively induced Abeta sequestration without provoking neuroinflammation; thus, these deglycosylated antibodies may be optimal for sequestration therapy for Alzheimer's disease.
Synthesis of Folate-conjugated Amphiphiles for Tumor-targeted Drug Delivery
Folic acid was derivatized specifically at its gamma-carboxyl group to retain its ligand-binding activity to the folate receptor alpha (FRalpha) present on HeLa cells. Amphiphilic molecules labeled with folic acid were prepared by conjugation of long-chain primary amines directly or via diamine linkers to the gamma-carboxyl group of folic acid. Folic acid amphiphiles labeled with fluorescent 7-amino-4-carbamoylmethylcoumarin (ACC) were also prepared to visualize the uptake of amphiphiles in folate receptor positive cells. The structures of the new compounds were verified by proton NMR and MALDI-TOF mass spectrometry. The amphiphiles form micelles in water. Critical micelle concentrations (CMCs) were determined by the pyrene fluorescence method for folic acid amphiphiles and the rise in capillary height method for the fluorescently labeled amphiphile. The CMCs of the amphiphiles were studied in buffer solution at pH 8 and ranged from 2 to 64 microM. The formation of micelle increased the solubility of paclitaxel, a model lipophilic anticancer compound, by more than 80%. A significant amount of the fluorescently tagged amphiphile was internalized into HeLa cells known to express FRsalpha when compared with Caco-2 cells that do not express FRalpha. Therefore, folate-labeled amphiphiles show promise in targeting antitumor agents to FRalpha-expressing cancer cells.
Expression and Characterization of Recombinant Human Secretory Leukocyte Protease Inhibitor (SLPI) Protein from Pichia Pastoris
The human secretory leukocyte protease inhibitor (SLPI) has been shown to possess anti-protease, anti-inflammatory and antimicrobial properties. Its presence in saliva is believed to be a major deterrent to oral transmission of human immunodeficiency virus-1. The 11.7kDa peptide is a secreted, nonglycosylated protein rich in disulfide bonds. Currently, recombinant SLPI is only available as an expensive bacterial expression product. We have investigated the utility of the methylotrophic yeast Pichia pastoris to produce and secrete SLPI with C-terminal c-myc and polyhistidine tags. The post-transformational vector amplification protocol was used to isolate strains with increased copy number, and culturing parameters were varied to optimize SLPI expression. Modification of the purification procedure allowed the secreted, recombinant protein to be isolated from the cell-free fermentation medium with cobalt affinity chromatography. This yeast-derived SLPI was shown to have an anti-protease activity comparable to the commercially available bacterial product. Thus, P. pastoris provides an efficient, cost-effective system for producing SLPI for structure function analysis studies as well as a wide array of potential therapeutic applications.
Gas-phase Scrambling of Disulfide Bonds During Matrix-assisted Laser Desorption/ionization Mass Spectrometry Analysis
Evidence for photo-induced radical disulfide bond scrambling in the gas phase during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described. The phenomenon was observed during the analysis of tryptic peptides from insulin and was confirmed in the determination of disulfide bonds in the rhamnose-binding lectin SEL24K from the Chinook salmon Oncorhynchus tshawytscha. A possible mechanism for this surprising scrambling is proposed. Despite this finding, the disulfide bond pattern in SEL24K was assigned unambiguously by a multi-enzyme digestion strategy in combination with MALDI mass spectrometry. The pattern was found to be symmetrical in the tandem repeat sequence of SEL24K. To the best of our knowledge, this is the first report of disulfide bond scrambling in the gas phase during MALDI-MS analysis. This observation has important ramifications for unambiguous assignment of disulfide bonds.
Secretion and Proteolysis of Heterologous Proteins Fused to the Escherichia Coli Maltose Binding Protein in Pichia Pastoris
The Escherichia coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N-terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris, a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N-terminal partner to several C-terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C-terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C-terminal of the MBP domain, including the spacer and cargo regions, but the MBP domain could still act to enhance the secretion of certain cargo proteins.
An Improved Method for Enhanced Production and Biological Activity of Human Secretory Leukocyte Protease Inhibitor (SLPI) in Pichia Pastoris
The human secretory leukocyte protease inhibitor (SLPI) is an 11.7 kD cysteine-rich protein that has been shown to possess anti-protease, anti-inflammatory, and antimicrobial properties. By using a Pichia pastoris strain that overproduces protein disulfide isomerase (PDI), we obtained greater than fivefold higher levels of SLPI than in strains expressing normal levels of PDI and containing multiple copies of the SLPI gene. Elevated levels of PDI also enhanced the specific activity of the secreted SLPI by helping it achieve a proper tertiary structure. Mass spectrometry analysis indicated a greater number of disulfide bonds in the SLPI produced by the PDI overexpression strain compared to the SLPI produced in strains with normal PDI levels. Although others have utilized a similar strategy to increase yield, we believe that this is the first example of PDI overexpression being demonstrated to enhance the folding and thus increase the biological activity of a protein produced in the yeast P. pastoris.
Synthetic Spider Silk Fibers Spun from Pyriform Spidroin 2, a Glue Silk Protein Discovered in Orb-weaving Spider Attachment Discs
Spider attachment disc silk fibers are spun into a viscous liquid that rapidly solidifies, gluing dragline silk fibers to substrates for locomotion or web construction. Here we report the identification and artificial spinning of a novel attachment disc glue silk fibroin, Pyriform Spidroin 2 (PySp2), from the golden orb weaver Nephila clavipes . MS studies support PySp2 is a constituent of the pyriform gland that is spun into attachment discs. Analysis of the PySp2 protein architecture reveals sequence divergence relative to the other silk family members, including the cob weaver glue silk fibroin PySp1. PySp2 contains internal block repeats that consist of two subrepeat units: one dominated by Ser, Gln, and Ala and the other Pro-rich. Artificial spinning of recombinant PySp2 truncations shows that the Ser-Gln-Ala-rich subrepeat is sufficient for the assembly of polymeric subunits and subsequent fiber formation. These studies support that both orb- and cob-weaving spiders have evolved highly polar block-repeat sequences with the ability to self-assemble into fibers, suggesting a strategy to allow fiber fabrication in the liquid environment of the attachment discs.
Analysis of the 5' Untranslated Region (5'UTR) of the Alcohol Oxidase 1 (AOX1) Gene in Recombinant Protein Expression in Pichia Pastoris
Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express over one thousand heterologous proteins valued for industrial, pharmaceutical and basic research purposes. In most cases, the 5' untranslated region (UTR) of the alcohol oxidase 1 (AOX1) gene is fused to the coding sequence of the recombinant gene for protein expression in this yeast. Because the effect of the AOX1 5'UTR on protein expression is not known, site-directed mutagenesis was performed in order to decrease or increase the length of this region. Both of these types of changes were shown to affect translational efficiency, not transcript stability. While increasing the length of the 5'UTR clearly decreased expression of a β-galactosidase reporter in a proportional manner, a deletion analysis demonstrated that the AOX1 5'UTR contains a complex mixture of both positive and negative cis-acting elements, suggesting that the construction of a synthetic 5'UTR optimized for a higher level of expression may be challenging.
