15N labeled VR18 was synthesized by Fmoc solid-phase peptide synthesis chemistry, as described previously, using an automated peptide synthesizer (Aapptec Endeavor 90, Louisville, KY, USA).13 (link), 14 (link) The Val-1, Ala-2, Gly-4, Gly-6, Leu-11 and Gly-13 residues were selectively labeled with 15N by using the 15N labeled Fmoc-amino acids, respectively. N-terminal Val-1 residue was subjected to acetylation in order to provide protection during the peptide cleavage step. The crude peptide was purified by a reverse-phase high performance liquid chromatographic system (RP-HPLC) (LC-20AT, SHIMADZU, Kyoto, Japan) using a Phenomenix C18 column. The peptide fraction was collected from the RP-HPLC elution and subjected to lyophilization to obtain the powder form. The calculated and actual molecular weight obtained for the peptides was 2107.49 and 2107.26, respectively. The purified peptide (>95% purity) was stored at −20°C.
Endeavor 90
Manufactured by AAPPTec
Sourced in United States
The Endeavor 90 is a high-performance automated peptide synthesizer designed for efficient and reliable synthesis of peptides. It features a compact design, intuitive user interface, and advanced software control to streamline the peptide synthesis process.
Automatically generated - may contain errors
Lab products found in correlation
Lc 20at, by Shimadzu (1 mentions)
Reverse phase hplc, by Shimadzu (1 mentions)
Hexafluoroisopropanol, by Merck Group (1 mentions)
Hiapp, by Merck Group (1 mentions)
Deuterium oxide d2o, by Cambridge Isotopes (1 mentions)
4 4 dimethyl 4 silapentane 1 sulfonic acid dss, by Cambridge Isotopes (1 mentions)
Thioflavint dye, by Merck Group (1 mentions)
Maldi tof, by Bruker (1 mentions)
Reverse phase hplc system, by Shimadzu (1 mentions)
Maldi tof mass spectrometry, by Bruker (1 mentions)
C18 column, by Waters Corporation (1 mentions)
7 protocols using endeavor 90
1
Synthesis and Purification of 15N-labeled VR18 Peptide
2
Solid-phase Synthesis of Insulin Peptides
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The four peptides were synthesized in solid-state peptide synthesizer from Aapptec endeavor 90. All the amino acids are F-moc linked. PyBOP was used as an activator of the reaction during peptide synthesis. We used DIPEA (N,N-diisopropylethylamine) as a base of the reaction. Also, the coupling reagent was DMF (N,N-dimethylformamide) and the de-coupling reagent was 20% piperidine. All the four peptides were synthesized and to provide stability to these peptides during synthesis it was acetylated at the end of the synthesis. The synthesized peptides were cleaved from the resin and side chain blockers by cleaving with trifluoroacetic acetic acid, anisol, phenol, tri-isopropanol saline. Then all the peptides were purified by reverse-phase HPLC. The purified peptides were further used to study insulin fibrillation.
3
Selective Isotope-Labeled Peptide Synthesis
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Selectively 15N-labeled NR25 (N45IV NV SLV KPSFYV YSRV KNL NSSR69-NH2) (the underlined amino acids are 15N labeled) peptide was synthesized on a solid phase peptide synthesizer (Aapptec Endeavor 90, USA) with 15N-labeled Fmoc protected Val and Leu. A solid-phase peptide synthesis protocol was used with a Rink Amide MBHA resin (substitution 0.69 mmol/g; Novabiochem, San Diego, California, USA) [19 (link)]. The resultant synthesized peptide was then purified using reverse-phase HPLC (SHIMADZU, Japan) on a Phenomenix C18 column (dimension 250 × 10 mm, with a pore size of 100 Å, 5-μm particle size). Linear gradient elution technique was employed with water and methanol along with 0.1% TFA (serving as the ion-pairing agent) as the solvent. Mass spectrometry and NMR spectroscopy were used to check the purity of the sample. The purified peptide was then shelved at −20 °C. Working stock solutions of 1 mM peptide was prepared either in sterile water or 10 mM phosphate buffer (pH 7.4) and used for the respective experiments and stored at 4 °C was short periods.
4
Solid-Phase Synthesis and Purification of Peptides
5
Synthesis and Purification of TK9 Peptide
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The TK9 peptide was synthesized in a semi-automated peptide synthesizer (Aapptec Endeavor 90) using Fluorenylmethyloxycarbonyl (Fmoc) protected amino acids (Novabiochem) and Rink Amide MBHA resin as described elsewhere [14] , [15] (link). The crude peptide was purified by High-performance liquid chromatography (HPLC) using a SHIMADZU (Japan) instrument with a Phenomenix C18 column (dimension 250 × 10 mm, pore size 100 Å, 5-μm particle size) and linear gradient elution. The ratio of methanol: water was varied linearly from 0 to 100% during the elution and the eluent fractions were lyophilized. Mass spectroscopy and NMR were used to confirm the purity and molecular weight of the eluted peptides.
6
Fmoc-based Peptide Synthesis and Purification
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Peptides (Table 1 ) are synthesized in Solid phase Peptide synthesizer (Aapptec Endeavor 90) using the principles of Fmoc chemistry. Fmoc-protected amino acids are sequentially coupled followed by fmoc deprotection using 20% piperidine solution for 60 and 40 min, respectively. DIPEA and PyBOP are used as activator base and activator respectively. After washing in DMF, peptide-attached resin is cleaved by standard resin cleavage cocktail solution containing 92.5% TFA, 2.5% milliQ water, 2.5% TIS, and 2.5% phenol. The cleaved filtrates are precipitated using diethyl ether solvent and purified using reverse phase HPLC system (SHIMADZU, Japan) with Phenomenix C18 column. Peptide masses are determined by MALDI-TOF mass spectrometry (Bruker). (Supplementary information Section 5 ).
7
Synthesis and Labeling of Antimicrobial Peptides
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Automated synthesis of three peptides derived from hBD3 (Figure 1 ) was carried out on a midi-scale peptide synthesizer (Endeavor 90, AAPP TEC, USA) to yield the C-terminal amide form using standard 9-fluorenylmethoxycarbonyl chemistry. Peptides were purified by preparative reverse-phase high-performance liquid chromatography (Waters, USA), with a Vydac C18 column and 50-minute gradient from 90% to 10% water/acetonitrile containing 0.1% trifluoroacetic acid. The purity of the synthetic peptides was determined by high-performance liquid chromatography analysis or liquid chromatography–mass spectrometry analysis. All purities were >90%. To detect the translocation pathways of the peptides, the synthetic peptides were manually conjugated at the N-terminus with the red dye rhodamine B during synthesis.12 (link) The purity and efficiency of rhodamine labeling were investigated via high-performance liquid chromatography by monitoring the absorbance at 230 nm and fluorescence at 560 nm.
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