Synthesis and Characterization of a Collagen Model δ-O-Phosphohydroxylysine-Containing Peptide

Hubálek, František; Edmondson, Dale E.; Pohl, Jan

doi:10.1006/abio.2002.5693

Cited by 19 publications

(24 citation statements)

References 43 publications

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“…28 The polypeptides yielded major single peaks eluting at 25-30% of acetonitrile and their masses were confirmed by MALDI-TOF MS or ESI-MS (see Table I and Results section).…”

Section: Mass Spectrometry Analysismentioning

confidence: 83%

“…Upon tryptic digestion at pH 6, rSD1 yielded a disulfide pattern identical to the one found for native SVN and SD1B. The m/z ¼ 2529.0 species, as well as its thermolytic fragment, residues [25][26][27][28][29][30]-[37-43] (m/z ¼ 1444.6), were sequenced. Under these conditions, the heterodimer of m/z ¼ 2511.0 was not found in the digest.…”

Section: Recombinant Sd1 Domain Assumes Natural Svn Foldmentioning

confidence: 97%

“…28 Electrospray ionization mass spectrometric analysis was performed in a Bruker Daltonics model maXis ESI-Q-TOF instrument interfaced either by flow-injection analysis using a syringe pump or LC-MS/MS using a Dionex model U3000 HPLC configured for nL/min flows. Flow-injection analysis was performed using a syringe pump flowing at 5 lL/min into a software controlled injection valve fitted with a 5 lL sample loop.…”

Section: Mass Spectrometry Analysismentioning

confidence: 99%

“…28,29 For pH 6 SVN digest, gradient slope of 0.6%/min of acetonitrile was used, 28,29 whereas gradient slope of 0.5%/min was used for a more complex, pH 8 digest to improve separation of the peptides. The peptides absorbing at 214 nm were manually collected and subjected to MALDI-TOF MS or to flow injection analysis on ESI-Q-TOF MS, to determine their monoisotopic masses, and to Edman degradation to determine their N-terminal sequences.…”

Section: Microbore Rp-hplc Of Svn Digestsmentioning

confidence: 99%

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Topology of the disulfide bonds in the antiviral lectin scytovirin

et al. 2010

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The antiviral lectin scytovirin (SVN) contains a total of five disulfide bonds in two structurally similar domains. Previous reports provided contradictory results on the disulfide pairing in each individual domain, and we have now re-examined the disulfide topology. N-terminal sequencing and mass spectrometry were used to analyze proteolytic fragments of native SVN obtained at acidic pH, yielding the assignment as Cys7-Cys55, Cys20-Cys32, Cys26-Cys38, Cys68-Cys80, and Cys74-Cys86. We also analyzed the N-terminal domain of SVN (SD1, residues 1-48) prepared by expression/oxidative folding of the recombinant protein and by chemical synthesis. The disulfide pairing in the chemically synthesized SD1 was forced into predetermined topologies: SD1A (Cys20-Cys26, Cys32-Cys38) or SD1B (Cys20-Cys32, Cys26-Cys38). The topology of native SVN was found to be in agreement with the SD1B and the one determined for the recombinant SD1 domain. Although the two synthetic forms of SD1 were distinct when subjected to chromatography, their antiviral properties were indistinguishable, having low nM activity against HIV. Tryptic fragments, the ''cystine clusters'' [Cys20-Cys32/Cys26-Cys38; SD1] and [Cys68-Cys80/ Disclaimer: The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.Abbreviations: Acm, acetamidomethyl; ATZ, 2-anilino-5-thiazolinone; AUFS, absorbance unit full scale; Boc-Gly-OCH 2 -PAM, t-butyloxycarbonyl-glycyl-4-(hydroxymethyl)phenylacetamidomethyl-copoly(styrene/1% divinylbenzene) resin; DIEA, diisopropylethylamine; ESI-Q-TOF, electrospray ionization quadrupole-time-of-flight mass spectrometry; HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino) methylene]-N-methyl-methanaminium hexafluorophosphate N-oxide; HFBA, heptafluorobutyric acid; MALDI-TOF, matrix-assisted laser desorption-ionization time-of-flight; MS/MS, tandem mass spectrometry; m/z, mass-to-charge ratio; pE, pyroglutamyl; PTH, phenylthiohydantoin; RP-HPLC, reversed-phase high pressure liquid chromatography; rSD1, recombinant/oxidatively folded N-terminal domain of SVN (residues 1-48Cys7Ser); R.T., retention time on HPLC; SD1A, synthetic N-terminal domain of SVN [residues 1-48Cys7Ser (Cys20-Cys26, Cys32-Cys38)]; SD1B, synthetic N-terminal domain of SVN [residues 1-48Cys7Ser (Cys20-Cys32, Cys26-Cys38)]; TCEP, tris(2-Carboxyethyl)phosphine; TFA, trifluoroacetic acid. Cys74-C-86; SD2], were found to undergo rapid disulfide interchange at pH 8. This interchange resulted in accumulation of artifactual fragments in alkaline pH digests that are structurally unrelated to the original topology, providing a rational explanation for the differences between the topology reported herein and the one reported earlier (Bokesh et al., Biochemistry 2003;42:2578-2584. Our observations emphasize the fact that proteins such as SVN, with disulfide bonds in close proximity, require consider...

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“…28 The polypeptides yielded major single peaks eluting at 25-30% of acetonitrile and their masses were confirmed by MALDI-TOF MS or ESI-MS (see Table I and Results section).…”

Section: Mass Spectrometry Analysismentioning

confidence: 83%

Section: Recombinant Sd1 Domain Assumes Natural Svn Foldmentioning

confidence: 97%

Section: Mass Spectrometry Analysismentioning

confidence: 99%

Section: Microbore Rp-hplc Of Svn Digestsmentioning

confidence: 99%

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Topology of the disulfide bonds in the antiviral lectin scytovirin

et al. 2010

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“…In the first, samples were microdesalted using a C18 ZipTip (Millipore, Bedford, MA, USA) and analyzed by MALDI-TOF/TOF MS/MS using a model 4700 Proteomics Analyzer (Applied Biosystems). The other method involved analysis by delayed-extraction reflectron MALDI-TOF-MS (ReflexIII mass spectrometer; Bruker Daltonics, Billarica, MA, USA), as described by Hubalek et al [24]. For each method, an MS spectrum was initially collected, and up to ten precursor molecular ions observed in each fraction were selected for subsequent MS/MS analysis.…”

Section: Protein Identification By Peptide Maldi-tof/ Tof Ms/ms and Mmentioning

confidence: 99%

Proteomic analysis of cerebrospinal fluid discriminates malignant and nonmalignant disease of the central nervous system and identifies specific protein markers

et al. 2006

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CNS diseases are often accompanied by changes in the protein composition of cerebrospinal fluid (CSF). SELDI-TOF-MS provides an approach for identifying specific protein markers of disease in biological fluids. We compared the CSF proteomes from patients with neoplastic and reactive/inflammatory CNS diseases to identify potential biomarkers. SELDI-TOF-MS was performed on CSF derived from lumbar puncture of 32 patients, including 10 with CNS malignancies, 12 with inflammatory or reactive conditions, and 10 with unknown CNS disease. Using the SAX-2 (strong anionic exchange) chip, we uncovered three conserved protein peak ranges within each disease category. For neoplastic diseases, we identified conserved peaks at 7.5-8.0 kDa (9/10 samples), 15.1-15.9 kDa (8/10 samples), and 30.0-32.0 kDa (5/10 samples). In reactive/inflammatory diseases, conserved peaks were found at 6.7-7.1 kDa (10/12 samples), 11.5-11.9 kDa (12/12 samples), and 13.3-13.7 kDa (9/12 samples). A protein from the 30.0 to 32.0 kDa peak range found in neoplastic CSF was identified by MALDI analysis as carbonic anhydrase, a protein overexpressed in many malignancies including high-grade gliomas. Similarly, cystatin C was identified in the 13.3-13.7 kDa peak range in non-neoplastic CSF and was most prominent in inflammatory conditions. Our approach provides a rational basis for identifying biomarkers that could be used for detection, diagnosis, and monitoring of CNS diseases.

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Eine Studie zur Ermittlung der Querkrafttragfähigkeit älterer Spannbetonbrücken auf Basis von Versuchsergebnissen

Strauss

2016

Beton und Stahlbetonbau

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In der vorgestellten Studie zur Ermittlung der Querkrafttragfähigkeit älterer parallelgurtiger Spannbetonbrücken werden die aus Querkraftversuchen resultierenden cot θ‐Werte mit den normgemäßen Werten und den an einem alternativen Rechenmodell ermittelten cot θ‐Werten verglichen. Die Ergebnisse der Studie gelten nur für den Sonderfall einer senkrechten Querkraftbewehrung (α = 90°). A study to determine the shear capacity of older prestressed concrete bridges based on test results In the presented study to determine the shear resistance of older prestressed concrete bridges with parallel girders the cot θ values resulting from shear experiments will be compared with the standard values and values determined on an alternative computing model. The results of the study apply only to the special case of a vertical shear reinforcement (α = 90°).

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Synthesis and Characterization of a Collagen Model δ-O-Phosphohydroxylysine-Containing Peptide

Cited by 19 publications

References 43 publications

Topology of the disulfide bonds in the antiviral lectin scytovirin

Topology of the disulfide bonds in the antiviral lectin scytovirin

Proteomic analysis of cerebrospinal fluid discriminates malignant and nonmalignant disease of the central nervous system and identifies specific protein markers

Eine Studie zur Ermittlung der Querkrafttragfähigkeit älterer Spannbetonbrücken auf Basis von Versuchsergebnissen

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