11 research outputs found
Representative annotated MS3 spectrum of the Pept+HexNAc product ion (m/z 1411, z=2).
<p>The Pept+HexNAc product ion derives from the MS2 fragmentation of the MH<sup>3+</sup> ion (m/z 1210) of Pept<sub>127-145</sub> glycoform B. The corresponding MS3 spectra for the other glycoforms were identical. The fragment ions with a <i>square</i> symbol are those with the HexNAc residue still in place, while those with a <i>star</i> have undergone the neutral loss of the HexNAc residue. HexNAc was annotated here at N<sub>131</sub> based on evidence not related to this MS3 spectrum (see main text). Annotated fragments are within 0.6 Da from the theoretical value.</p
Ī³-Conglutin glycoform profile obtained by LC-Orbitrap MS analysis of the in-gel tryptic digest of the large subunit.
<p>The MS spectrum (averaged over the elution time of the A-E and Bā²-Eā² glycoforms of Pept<sub>127-165</sub>, Pept<sub>111-165</sub>, and the non-glycosylated Pept<sub>127-165</sub>) was charge-deconvoluted and deisotoped to show the monoisotopic MH<sup>+</sup> ions.</p
Schematic overview of the glycoproteomic workflow.
<p>Combinations of the following procedures were used: 1) reducing SDS-PAGE to isolate the N-glycosylated large subunit, 2) Ī³-conglutin proteolytic digestion in-gel (with trypsin) or in-solution (with endopeptidase GluC (V8) followed by trypsin) to generate different mixtures of peptides and N-glycopeptides; 3) N-deglycosylation of the digests with PNGase A or PNGase F to identify the N-glycosylation site, and assess the presence/absence of ācoreā Ī±1-3 fucose. The digests (with or without N-deglycosylation) were then analyzed by LCāOrbitrap MS, with MS survey scans followed by data-dependent ITMS2 or targeted MSn. Mass spectral data analysis included the use of: 1) automated charge-deconvolution of high resolution-high mass accuracy spectra, and isotope envelope simulation; 2) bioinformatic tools for <i>in </i><i>silico</i> glycoform structure prediction (GlycoMod and GlycoSuiteDB), and database search (Mascot) for sequence identification of non-glycosylated or enzymatically deglycosylated peptides; (3) manual inspection of MS2 and MS3 spectra of glycopeptides for sequence annotation of their monosaccharide and amino acid components, respectively.</p
Annotated MS2 mass spectra of the A to E glycoforms of Pept<sub>122-145</sub>.
<p>The MS2 mass spectra were obtained by fragmenting MH<sup>3+</sup> ions at 1165, 1209, 1263, 1331 and 1399 m/z (A to E glycoforms respectively). The dashed lines connect fragment ions with identical m/z values across the MS2 spectra. The fragments are annotated at their first appearance only (top to bottom), following the Consortium for Functional Glycomics Symbol Nomenclature. Braces within the glycan structure refer to possible alternative linkages (see Table S1).</p
Proposed N-glycan structures for Ī³-conglutin glycoforms A to E.
<p>Structures are drawn using the Consortium for Functional Glycomics Symbol Nomenclature (<a href="http://www.functionalglycomics.org/static/consortium/nomenclature.shtml" target="_blank">http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml</a>), with GlcNAc=square; Fuc=triangle; Man=circle; Xyl=five-pointed star. Braces within the glycan structure indicate possible alternative linkages. For linkage details, see Table S1.</p
Access to Ī±,Ī±-Disubstituted Disilylated Amino Acids and Their Use in Solid-Phase Peptide Synthesis
A concise synthetic pathway yielding
to hydrophobic Ī±,Ī±-disubstituted
disilylated amino acids based on a hydrosilylation reaction is described.
As a first example of utilization in solid-phase peptide synthesis,
TESDpg was introduced in replacement of Aib in an alamethicin sequence,
leading to analogues with modified physicochemical properties and
conserved helical structures. This study highlights the potential
of these new amino acids as tools for peptide modulation
Hydrophobic Ī±,Ī±-Disubstituted Disilylated TESDpg Induces Incipient 3<sub>10</sub>-Helix in Short Tripeptide Sequence
To evaluate the contribution
of triethylsilyl Ī±,Ī±-di-<i>n</i>-propylglycine,
namely TESDpg, to induce a defined secondary
structure, we have prepared model tripeptides in which TESDpg was
inserted in three different positions. Studies in solid state and
in solution with adapted techniques showed that TESDpg was able to
induce a nascent 3<sub>10</sub> helix in both crystal and solution
states
Use of Molecular Modeling to Design Selective NTS2 Neurotensin Analogues
Neurotensin exerts
potent analgesia by acting at both NTS1 and
NTS2 receptors, whereas NTS1 activation also results in other physiological
effects such as hypotension and hypothermia. Here, we used molecular
modeling approach to design highly selective NTS2 ligands by investigating
the docking of novel NTĀ[8-13] compounds at both NTS1 and NTS2 sites.
Molecular dynamics simulations revealed an interaction of the Tyr<sup>11</sup> residue of NTĀ[8-13] with an acidic residue (Glu<sup>179</sup>) located in the ECL2 of hNTS2 or with a basic residue (Arg<sup>212</sup>) at the same position in hNTS1. The importance of the residue at
position 11 for NTS1/NTS2 selectivity was further demonstrated by
the design of new NT analogues bearing basic (Lys, Orn) or acid (Asp
or Glu) function. As predicted by the molecular dynamics simulations,
binding of NTĀ[8-13] analogues harboring a Lys<sup>11</sup> exhibited
higher affinity toward the hNTS1-R212E mutant receptor, in which Arg212
was substituted by the negatively charged Glu residue
Use of Molecular Modeling to Design Selective NTS2 Neurotensin Analogues
Neurotensin exerts
potent analgesia by acting at both NTS1 and
NTS2 receptors, whereas NTS1 activation also results in other physiological
effects such as hypotension and hypothermia. Here, we used molecular
modeling approach to design highly selective NTS2 ligands by investigating
the docking of novel NTĀ[8-13] compounds at both NTS1 and NTS2 sites.
Molecular dynamics simulations revealed an interaction of the Tyr<sup>11</sup> residue of NTĀ[8-13] with an acidic residue (Glu<sup>179</sup>) located in the ECL2 of hNTS2 or with a basic residue (Arg<sup>212</sup>) at the same position in hNTS1. The importance of the residue at
position 11 for NTS1/NTS2 selectivity was further demonstrated by
the design of new NT analogues bearing basic (Lys, Orn) or acid (Asp
or Glu) function. As predicted by the molecular dynamics simulations,
binding of NTĀ[8-13] analogues harboring a Lys<sup>11</sup> exhibited
higher affinity toward the hNTS1-R212E mutant receptor, in which Arg212
was substituted by the negatively charged Glu residue
Synthesis and Characterization in Vitro and in Vivo of (l)ā(Trimethylsilyl)alanine Containing Neurotensin Analogues
The
silylated amino acid (l)-(trimethylsilyl)Āalanine (TMSAla)
was incorporated at the C-terminal end of the minimal biologically
active neurotensin (NT) fragment, leading to the synthesis of new
hexapeptide NT[8ā13] analogues. Here, we assessed the ability
of these new silylated NT compounds to bind to NTS1 and NTS2 receptors,
promote regulation of multiple signaling pathways, induce inhibition
of the ileal smooth muscle contractions, and affect distinct physiological
variables, including blood pressure and pain sensation. Among the
C-terminal modified analogues, compound <b>6</b> (JMV2007) carrying
a TMSAla residue in position 13 exhibits a higher affinity toward
NT receptors than the NT native peptide. We also found that compound <b>6</b> is effective in reversing carbachol-induced contraction
in the isolated strip preparation assay and at inducing a drop in
blood pressure. Finally, compound <b>6</b> produces potent analgesia
in experimental models of acute and persistent pain