A further 10 mg of lyophilised skin secretion were dissolved in 1.5 mL of 0.05/99.95 (v/v) trifluoroacetic acid (TFA) /water and then clarified by centrifugation. The supernatant (1 mL) was then subjected to reversed phase HPLC using a Waters gradient reversed phase HPLC system, fitted with an analytical column (Jupiter, C5, 300 Å, 5 μm, 4.6 mm × 250 mm, Phenomenex, Macclesfield, Cheshire, UK). Column elution was achieved with a gradient formed from 0.05/99.95 (v/v) TFA/water to 0.05/29.95/70.00 (v/v/v) TFA/water/acetonitrile in 240 min at a flow rate of 1 mL/min, and the effluent was monitored by UV absorbance at 214 nm 280 nm. The eluted fractions were collected at 1 min intervals. The molecular masses of peptides in each fraction were further analysed by use of a MALDI-TOF mass spectrometer (Voyager DE, PerSeptive Biosystems, Foster City, CA, USA) in positive detection mode using α-Cyano-4-hydroxycinnamic Acid (CHCA) as the matrix. Fractions with peptide molecular masses coincident with those of the mature peptides predicted from the cloned cDNA, were then infused into an LCQ Fleet ion-trap electrospray mass spectrometer (Thermo Fisher Scientific, San Francisco, CA, USA) followed by trapping of appropriate ions for MS/MS fragmentation.
Jupiter c5
Manufactured by Phenomenex
Sourced in United Kingdom, United States
The Jupiter C5 is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a C5 stationary phase, which provides a moderate level of hydrophobicity for the retention of both polar and non-polar analytes.
Automatically generated - may contain errors
Lab products found in correlation
Lcq fleet electrospray ion trap mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Hplc system, by Waters Corporation (1 mentions)
Talon superflow metal affinity resin, by Takara Bio (1 mentions)
Lcq electrospray ion trap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Talon metal affinity resin, by Takara Bio (1 mentions)
Lcq fleet ion trap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Rp hplc system, by Waters Corporation (1 mentions)
10 protocols using jupiter c5
1
Peptide Isolation from Skin Secretion
2
Peptide Identification from Secretion Samples
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After centrifugation (2500×g , 5 min) from 10 mg/ml secretion solution dissolved in 0.05/99.5 (v/v) trifluoroacetic acid (TFA)/water, the clear supernatant was subjected to RP-HPLC column (Jupiter C5; 250 mm × 4.6 mm, Phenomenex, U.K.) on a Cecil CE4200 Adept gradient system (Cambridge, U.K.) (a gradient formed from 0.05/99.5 (v/v) TFA/water to 0.05/19.95/80.0 (v/v/v) TFA/water/acetonitrile in 240 min at a flowrate of 1 ml/min). Subsequently, fractions were taken every minute and each was analysed using MALDI-TOF mass spectrometer (α-cyano-4-hydroxycinnamic acid as the matrix) on a linear time-of-flight Voyager DE mass spectrometer (Perseptive Biosystems, MA, U.S.A.). The fractions with masses coincident with putative novel cDNA-encoded peptide were subjected to primary structural analysis by MS/MS fragmentation sequencing using an LCQ-Fleet electrospray ion-trap mass spectrometer (Thermo Fisher Scientific).
3
Purification and Characterization of Novel Peptide
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Five mg of lyophilised skin secretion were dissolved, clarified, and injected into an HPLC system (Waters, Milford, MA, USA) to separate the fractions using a gradient programme which ran over 240 min at a flow rate of 1 mL/min from water/TFA (99.95/0.05, v/v) to acetonitrile/water/TFA (80/19.95/0.05; v/v/v) on an analytical column (Jupiter C5, 5 μm, 240 mm × 4.6 mm, Phenomenex, Macclesfield, Cheshire, UK). The effluent was constantly monitored by a UV detector set at 214 nm (λ) and the fractions were automatically collected at minute intervals. All fractions were analysed by MALDI-TOF/MS (Voyager DE, Perspective Biosystems, Foster City, CA, USA) with CHCA as the matrix in positive mode. The instrument was calibrated by standards and set accuracy was ±0.1%. The peptide with a molecular mass coincident with that predicted from cloned cDNA, was injected into an LCQ-Fleet electrospray ion-trap mass spectrometer to analyse its primary structure by MS/MS fragmentation (Thermo Fisher Scientific, San Francisco, CA, USA).
4
Fractionation of T. oblongus Toxin
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ω-Tbo-IT1 toxin was fractionated from T. oblongus venom using a size-exclusion chromatography (SEC) and several steps of reverse-phase high pressure liquid chromatography (RP-HPLC). Soluble venom (2.7 mg in 5 ml) was applied to a Beckman TSK 2000SW column (7.5 × 600 mm) which was equilibrated with 20 mM sodium phosphate buffer (150 mM NaCl, pH 4.5). Separation was done at a flow rate of 0.5 ml/min, and chromatographic results were monitored at 214 nm. The active fraction was then applied on a Jupiter C5 (Phenomenex, USA) RP-HPLC column (4.6 × 250 mm) for separation in 0.1% v/v trifluoroacetic acid (TFA) buffer system in a linear gradient of acetonitrile concentration at a flow rate of 1 ml/min. The absorbance was monitored both at 210 nm and 280 nm. One active rough fraction was obtained as a result of the toxicity test on insects as described below. This active fraction was further fractionated on a Synergy Polar-RP400 (Phenomenex, USA) column (4.6 × 250 mm) in a linear gradient of acetonitrile (0.1% v/v TFA containing buffers) at a flow rate of 0.5 ml/min. Pure polypeptide elution time was monitored at the same wavelengths, 210 nm and 280 nm.
5
Peptide Purification from Skin Secretion
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A further five milligrams of lyophilized skin secretion were dissolved in 0.5 mL of 0.05% (v/v) trifluoroacetic acid (TFA)/water and clarified by centrifugation. The clear supernatant was subjected to reverse-phase HPLC on an analytical column (Jupiter C-5, 250 mm × 10 mm, Phenomenex, Macclesfield, Cheshire, UK), eluted with a 0%–80% linear gradient of acetonitrile containing 0.05% (v/v) TFA in 240 min at a flow rate of 1 mL/min. Absorbance was monitored at 214 nm. Fractions were collected at minute intervals and samples from each were analyzed by matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) (Perseptive Biosystems, Voyager DE, Perseptive Biosystems, Foster City, CA, USA). The fraction(s) containing peptides with molecular masse coincident with those calculated from cloned precursor-encoding cDNAs were then subjected to MS/MS fragmentation sequencing using an electrospray ion-trap mass spectrometer (Thermo Fisher Scientific, San Francisco, CA, USA).
6
Purification of Recombinant Polypeptides APHC1-3
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Polypeptides APHC1-3 were produced as previously described [24 (link)]. DNA fragments encoding the polypeptides were cloned into the expression vector pET32b+ (Novagen, USA). Escherichia coli BL21 (DE3) cells expressing thioredoxin fusions of polypeptides were cultured overnight at 25°C, harvested, ultrasonicated in a buffer for metal-affinity chromatography, and centrifuged to remove all insoluble particles. Fusion proteins were purified using a TALON Superflow Metal Affinity Resin (Clontech, USA) and subjected to CNBr cleavage, as described [30 (link)]. The recombinant polypeptides were purified on a reverse-phase column Jupiter C5 (Phenomenex, USA) 250×10 mm. The target polypeptides’ purity was verified by MALDI-TOF mass-spectrometry.
7
Peptide Mapping of Natural Skin Secretions
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A further five milligrams of lyophilised skin secretion were dissolved in 0.5 ml of 0.05% (v/v) trifluoroacetic acid (TFA)/water and clarified by centrifugation. The clear supernatant was subjected to reverse-phase HPLC on an analytical column (Jupiter C-5, 250 mm ×10 mm; Phenomenex, Cheshire, UK), eluted with a 0–80% linear gradient of acetonitrile containing 0.05% (v/v) TFA in 240 min at a flow rate of 1 ml/min. Absorbance was monitored at 214 nm. The peptide mapping of two natural occurring peptides was conducted using MS/MS fragmentation sequencing against the cDNA encoding peptide precursors by LCQ electrospray ion-trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).
8
Isolation and Bioactivity Screening of Skin Secretions
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Five milligrams of lyophilized skin secretion were dissolved in 1 mL of 0.05/99.95 (v/v) trifluoroacetic acid (TFA)/water and clarified by centrifugation. The supernatant was directly injected onto a CECIL reverse phase HPLC system (Milton Technical Centre, Cambridge, UK) fitted with an analytical column (Phenomenex Jupiter C5; 300 Å pore size; 250 × 4 mm). The linear elution gradient employed was formed from 0.05/99.95 (v/v) TFA/water to 0.05/19.95/80.0 (v/v/v) TFA/water/acetonitrile in 80 min at a flow rate of 1 mL/min. The effluent absorbance was monitored at λ = 214 nm and fractions of approximately 1 mL were collected automatically at minute intervals. Samples (100 μL) from each chromatographic fraction were removed, lyophilized, and stored at −20°C prior to analysis for myoactivity using rat smooth muscle bioassays.
9
Recombinant Protein Purification Protocol
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E. coli BL21(DE3) cells were transformed with expression vectors by electroporation. Transformed cells were cultured in LB medium with ampicillin (100 mg/ml) at 37 °C up to solution density 0.6–0.7 AU at 600 nm. T7-promoted expression was induced by addition of isopropyl-1-thio-β-D-galactopyranoside (IPTG) up to 0.2 mM concentration. Expression was performed for 12 h at 24 °C, after which cells were harvested and re-suspended in chromatogram buffer (300 mM NaCl, 20 mM Tris-HCl pH 7.5). The suspension was ultrasonicated, and the supernatant, after centrifugation (15 min at 14,000g), was applied to TALON Metal Affinity resin (Clontech Laboratories, USA). Fusion protein purification was made in accordance with the manufacturer’s manual. Protein cleavage was performed overnight at room temperature in the dark in accordance with method35 (link). HCl to a final concentration of 0.2 M and CNBr with a molar ratio to a fusion protein of 600:1 were added. Final purification of the recombinant polypeptide was made by RP-HPLC on Jupiter C5 (Phenomenex, USA) column, and then its identity to natural polypeptide was confirmed by MALDI-MS.
10
Skin Secretion Peptide Purification and Identification
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An aliquot of 5 mg of lyophilised skin secretions were dissolved in 1 mL of trifluoroacetic acid (TFA)/water (0.05/99.95, v/v) and clarified by centrifugation (2500× g for 5 min). The supernatant was then injected and pumped onto a RP-HPLC column (Jupiter, C5, 300 Å, 5 μm, 250 mm × 4.6 mm, Phenomenex, Macclesfield, Cheshire, UK) which was attached to a RP-HPLC system (Waters, Milford, MA, USA). The sample was eluted with a linear gradient from TFA/water (0.05/99.95; v/v) to TFA/water/acetonitrile (0.05/19.95/80.00; v/v/v) in 240 min at a flow rate of 1 mL/min, and all fractions were collected at one minute intervals. The collected fractions were analysed by a Voyager DE MALDI-TOF MS (Perseptive Biosystems, Bedford, MA, USA) in a positive detection mode using α-cyano-4-hydroxycinnamic acid matrix. The fraction containing the peptide with a molecular mass similar to the putative cDNA-encoding peptide was analysed by tandem mass (MS/MS) fragmentation using an LCQ-Fleet ion-trap mass spectrometer (ThermoFisher Scientific, San Francisco, CA, USA).
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