All chemicals involved in synthesis or analysis were obtained from Aldrich (Milwaukee, WI, USA) or Novabiochem (San Diego, CA, USA) unless otherwise indicated. All reactions were performed at room temperature unless indicated otherwise. Analytical HPLC was performed on a HP1100 series instrument (Agilent, Santa Clara, CA, USA) with 220 and 280 nm detection using a Vydac C18 column (5 μm, 4.6 × 150 mm; Grace, Columbia, MD, USA) at a flow rate of 1 mL/min. Preparative and semipreparative HPLC were performed on a Waters Delta Prep system (Waters Co., Milford, MA, USA) fitted with a Waters 2487 UV-visible detector using either a Vydac C18 column (15–20 μm, 50 × 250 mm) or a Vydac C18 (15–20 μm, 10 × 250 mm) at a flow rate of 50 or 5 mL/min, respectively. All runs used linear gradients of 0.1% aqueous trifluoroacetic acid (TFA) (solvent A) vs. 0.1% TFA, 90% acetonitrile in H2O (solvent B). Ultraviolet-visible (UV-vis) spectroscopy was carried out on an Agilent 8453 diode array spectrophotometer (Agilent). Electrospray mass spectrometry (ES-MS) analysis was routinely applied to all compounds and components of reaction mixtures. ES-MS was performed on an Applied Biosystems API 3000 triple quadrupole electrospray mass spectrometer (Applied Biosystems, Foster City, CA, USA) using Analyst 1.4.2. Calculated masses were obtained using Analyst 1.4.2.
Vydac c18 column
Manufactured by Grace Bio-Labs
Sourced in United States
The Vydac C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and purification of a wide range of compounds. It features a stationary phase of octadecylsilyl (C18) bonded silica particles, providing efficient and reproducible chromatographic separations.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Hp1100 series instrument, by Agilent Technologies (2 mentions)
Waters delta prep system, by Waters Corporation (2 mentions)
Vydac c18 column, by Waters Corporation (2 mentions)
Vydac c18, by Waters Corporation (2 mentions)
Applied biosystems api 3000 triple quadrupole electrospray mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Agilent 8453 diode array spectrophotometer, by Agilent Technologies (2 mentions)
Waters 2487, by Waters Corporation (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Vydac c18 analytical column, by Grace Bio-Labs (1 mentions)
Masslynx software, by Waters Corporation (1 mentions)
Quadrupole mass spectrometer, by Agilent Technologies (1 mentions)
Lc msd system, by Agilent Technologies (1 mentions)
Esi interface, by Agilent Technologies (1 mentions)
Enterokinase, by New England Biolabs (1 mentions)
Acetylcholine chloride ach, by Merck Group (1 mentions)
Protein hispurtm ni nta purification kit, by Thermo Fisher Scientific (1 mentions)
Abi 4800 maldi tof tof system, by Thermo Fisher Scientific (1 mentions)
10 protocols using vydac c18 column
1
Analytical and Preparative HPLC Methods
2
Lyba1 Peptide Characterization and Sequencing
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Lyba1 was partially reduced in 100 µL of 50 mM Tris(2-carboxyethyl)phosphine (TCEP) and 10% ACN at 65 °C for 2 min. Subsequently, N-ethylmaleimide (NEM) was added to a final concentration of 250 mM and incubated at 37 °C for 1 h. The intermediates were then fractionated with an analytical RP-HPLC with a Vydac C18 column (particle size: 5 µm, 250 × 4.6 mm; Grace, Maryland, USA) at a flow rate of 0.3 mL/min with a gradient of 23–37% over 80 min with buffer A (0.1% TFA) and buffer B (100% ACN, 0.1% TFA). Intermediate species were collected and analyzed using MALDI-TOF MS. Each intermediate species was then fully reduced with 50 µL of 20 mM DTT, 25 mM ammonium bicarbonate in 20% (v/v) ACN at 37 °C for 1 h and alkylated with 40 mM iodoacetamide (IAA) at 37 °C for 2 h. Alkylated samples were then examined by MALDI-TOF/TOF and sequenced by tandem mass spectrometry.
3
Synthesis and Characterization of CSP1hβE Peptide
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The peptide CSP1hβE (sequence: H-hβGlu-Met-Arg-Leu-Ser-Lys-Phe-Phe-Arg-Asp-Phe-Ile-Leu-Gln-Arg-Lys-Lys-OH; Table 1 ) was synthesized on Fmoc-Lys(Boc) Wang resin (0.44 mmol/g) using Fmoc chemistry by a Syro I synthesizer (Multisyntech; Witten, Germany). The side-chain protecting groups were OtBu, Boc, Trt, Pbf, and tBu. The coupling details are reported for each step in Table 2 . The cleavage from the resin was carried out, as indicated in previous paragraphs. The identity of the peptide was determined by mass spectrometry (theoretical mass = 2256 Da; experimental mass = 2258.07 Da; SCIEX TOF-TOF 4800 instrument). The peptide CSP1hβE was purified by RP-HPLC (purity grade > 95%) and characterized by analytical RP-HPLC (condition: Vydac C18 column (5 μm, 300 Å, 4.6 × 205 mm, Grace); eluent A: 0.05% TFA in H2O; eluent B: 0.05% TFA in CH3CN; gradient: from 25 to 35% B over 20 min; flow rate: 1 mL/min; detector: 214 nm; tR = 9.483 min).
4
Synthesis and Characterization of CSP1Y(SO3) Peptide
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The peptide CSP1Y(SO3) (sequence: H-Tyr(SO3H)-Met-Arg-Leu-Ser-Lys-Phe-Phe-Arg-Asp-Phe-Ile-Leu-Gln-Arg-Lys-Lys-OH; see Table 1 ) was synthesized on Fmoc-Lys(Boc) Wang resin (0.44 mmol/g) using Fmoc chemistry by a Syro I synthesizer (Multisyntech; Witten, Germany). The side-chain protecting groups were OtBu, Boc, Trt, Pbf, and tBu. The coupling details are reported for each step in Table 2 . The cleavage from the resin was carried out as indicated in previous paragraphs. The identity of the crude peptide was determined by mass spectrometry (theoretical mass = 2363.84 Da; experimental mass = 2357.3 Da; ESI-TOF, Mariner System 5220, Applied Biosystem, Perkin-Elmer, Foser City, CA, USA). The peptide CSP1Y(SO3) was isolated by RP-HPLC (purity > 95%) and characterized by analytical RP-HPLC (condition: Vydac C18 column (5 μm, 300 Å, 4.6 × 205 mm, Grace); eluent A: 0.05% TFA in H2O; eluent B: 0.05% TFA in CH3CN; gradient: from 25 to 35% B over 20 min; flow rate: 1 mL/min; detector: 214 nm; tR = 11.827 min).
5
Synthesis of Methionine-containing Dicarba Peptide
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Dicarba peptide 5 (0.3 mg, 170 nmol) was dissolved in TFA (0.5 mL). To the peptide solution, 2-methyl-2-butene (11 µL, 103 µmol) and bromotrimethylsilane were added (26 µL, 197 µmol). The reaction was shaken at room temperature for 15 min before removing TFA under flow of N2. The remaining residue was taken up in ice-cold Et2O (1 mL), and the precipitated peptide was collected by centrifugation (1 × 1 min). RP-HPLC and mass spectral analysis of the peptide supported the formation of the methionine-containing dicarba peptide 6 in quantitative conversion as a mixture of E- and Z-isomers. RP-HPLC (Grace, Columbia, MD, USA, Vydac C18 analytical column, 5–35% buffer B over 30 min): tR = 11.4 and 11.6 min. Mass spectrum (ESI+, MeCN:H2O:TFA): m/z 875.3 [M + 2H]2+, (C69H103N23O25S3) theoretical 874.8. The reaction mixture was purified by analytical RP-HPLC (Grace, Columbia, MD, USA, Vydac C18 column, 5–35% buffer B over 30 min, tR = 11.4 and 11.6 min). Coeluting isomer fractions were combined and lyophilised to give the target peptide 6 a colourless solid (0.1 mg, 95% purity).
6
Analytical and Preparative HPLC Methods
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All chemicals involved in synthesis or analysis were obtained from Aldrich (Milwaukee, WI, USA) or Novabiochem (San Diego, CA, USA) unless otherwise indicated. All reactions were performed at room temperature unless indicated otherwise. Analytical HPLC was performed on a HP1100 series instrument (Agilent, Santa Clara, CA, USA) with 220 and 280 nm detection using a Vydac C18 column (5 μm, 4.6 × 150 mm; Grace, Columbia, MD, USA) at a flow rate of 1 mL/min. Preparative and semipreparative HPLC were performed on a Waters Delta Prep system (Waters Co., Milford, MA, USA) fitted with a Waters 2487 UV-visible detector using either a Vydac C18 column (15–20 μm, 50 × 250 mm) or a Vydac C18 (15–20 μm, 10 × 250 mm) at a flow rate of 50 or 5 mL/min, respectively. All runs used linear gradients of 0.1% aqueous trifluoroacetic acid (TFA) (solvent A) vs. 0.1% TFA, 90% acetonitrile in H2O (solvent B). Ultraviolet-visible (UV-vis) spectroscopy was carried out on an Agilent 8453 diode array spectrophotometer (Agilent). Electrospray mass spectrometry (ES-MS) analysis was routinely applied to all compounds and components of reaction mixtures. ES-MS was performed on an Applied Biosystems API 3000 triple quadrupole electrospray mass spectrometer (Applied Biosystems, Foster City, CA, USA) using Analyst 1.4.2. Calculated masses were obtained using Analyst 1.4.2.
7
Identification of Peptides in P. microps
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To confirm the actual secretion of predicted peptides in P. microps, 10 µL of diluted skin secretion sample was injected into a Waters Breeze analytical HPLC system coupled to a Micromass Q-Tof micro system. The HPLC is equipped with a Waters 2696 pump, Waters 2489 UV/visible detector (at a wavelength of 215 nm) and a Grace Vydac C18 column (15 cm × 2.1 mm × 3 µm) with a flow rate of 0.3 mL/min. Electrospray data were acquired by electrospray positive ionization mode (ESI+) scanning over the mass-to-charge ratio (m/z) scale from 100 to 3000 at a scan time of one second and a cone voltage of 38 V. Data collection and analysis were done with Masslynx software (Waters Co.).
8
Identification of Compounds via HPLC-ESI-MS/MS
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The HPLC-ESI-MS/MS analyses were performed using a 1100 Series Agilent Technologies LC/MSD system equipped with a diode array detector coupled to a quadrupole mass spectrometer with an ESI interface (Agilent Technologies, Mississagua, Ont.). The reverse-phase separation was performed on a Vydac C18 column (5 μm; 250 mm × 4.6 mm; Grace, Hespedia, CA.). The compounds were separated using eluent (A): 4.5% aqueous formic acid and eluent (B): acetonitrile diluted to 80% with 4.5% formic acid in water. The gradient was as follows: 7 min 15% B; 15 min 20% B; 16 min 100% B and in 24 min returned to 100% of eluent A. MS parameters were as follows: capillary voltage 4000 V; drying gas temperature 350 o C; nitrogen fl ow 12 L/min; nebulizer pressure 60 psi. The instrument was scanned positive and negative ions in the range from 100 to 1500 m/z at a rate of 2.0 s/cycle. Compounds identifi cation is based on spectra library developed in laboratory and published data (Gil-Izquierdo and Mallenthin, 2001) .
9
Purification and Expression of Recombinant Proteins
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The Escherichia coli strains DH5α and BL21(DE3) pLysS, as well as expression vector pET-32a (+), were purchased from Novagen (Darmstadt, Hesse, Germany). Restriction enzyme Kpn I, EcoR I and Mlu I, T4 DNA ligase, protein markers, protein HisPurTM Ni-NTA purification kit, Fetal Bovine serum (FBS), penicillin, streptomycin, and cRNA mMESSAGE mMACHINE In Vitro Transcription Kit were purchased from Thermo Scientific (Waltham, MA, USA). enterokinase was purchased from New England Biolabs, Inc. (Ipswich, MA, USA). Vydac C18 columns (5 μm, 4.6 mm × 250 mm, 10 μm, 22 mm × 250 mm) were purchased from Grace (Deerfield, IL, USA). Acetonitrile (ACN, gradient grade for HPLC), Acetylcholine chloride (ACh) and other chemical reagents were all of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).
10
HPLC Analysis and MALDI-TOF/TOF Mass Spectrometry
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High-performance liquid chromatography (HPLC) analysis was performed on a Shimadzu system (Kyoto, Japan). Vydac C18 columns (Grace, Columbia, MD, USA; particle size 5 μm, pore size 300 Å) with dimensions of 250 × 22 mm and 250 × 4.6 mm were used for preparative and analytical RP-HPLC, respectively. Mass spectrometry analysis was performed on the ABI 4800 MALDI-TOF/TOF system (Applied Biosystems, Waltham, MA, USA). Chemical reagents used in this study were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA).
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