The desalted peptide samples were fractionated by strong cation exchange (SCX) chromatography using an Agilent 1200 HPLC system equipped with a Phenomenex Luna SCX column. A binary linear gradient consisting of buffer A (5 mM KH2PO4, pH 2.6, 30% acetonitrile (ACN)) and buffer B (5mM KH2PO4, pH 2.6, 30% ACN, 350 mM KCl) was applied at a flow rate of 1.5 mL/min for 97.5 min as follows: 0% B at 0 min, 5% B at 7.5 min, 60% B at 47.5 min, 100% B at 67.5 min, 100% B at 77.5 min, 0% B at 77.51 min to completion. Fractions were taken every 5 min starting at 17.5 min, and fractions were pooled as follows: 1–5, 6–7, 8, 9, 10, 11–14. Fractions 1–5 were not processed any further. The remaining fractions were then reduced to a final volume of 1–2 mL by vacuum centrifugation and pH adjusted to a pH of 8.0 with 1.5M NaOH. After pH adjustment, each sample was incubated for 1 hour with 100 μL of UltraLink monomeric avidin (Thermofisher) with gentle agitation. The avidin matrix was washed 5 times after this incubation period using 3mL aliquots of 100 mM ammonium bicarbonate, and cross-linked peptides were then eluted off the avidin beads by two additions of 500 μL 70% acetonitrile-0.5% formic acid. The resulting eluent was concentrated by vacuum centrifugation.
Luna scx column
Manufactured by Phenomenex
The Luna SCX column is a strong cation exchange chromatography column designed for the separation and purification of charged analytes. It features a silica-based stationary phase with sulfonic acid functional groups that interact with positively charged molecules. The Luna SCX column is suitable for a variety of applications requiring cation exchange separation.
Automatically generated - may contain errors
Lab products found in correlation
Ultralink monomeric avidin, by Thermo Fisher Scientific (1 mentions)
1200 hplc system, by Phenomenex (1 mentions)
Agilent 6410 triple quadrupole lc ms, by Agilent Technologies (1 mentions)
Agilent 6410 triple quadrupole, by Agilent Technologies (1 mentions)
20ad hplc system, by Shimadzu (1 mentions)
6 protocols using luna scx column
1
SCX Fractionation and Avidin Enrichment
2
Enzymatic Assay for Hydroxyproline Metabolism
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Enzyme was activated as described in ‘End-point activity assays with HypD variants.’ Assays contained 0.8 mM NADH, 3 µM P5CR, 0.2 mM Hyp, and 0.3 µM HypD (HypD added as a mixture of activation components) in Tris buffer made with D2O (20 mM Tris-HCl pH 7.5, 100 mM KCl). Assays were carried out in triplicate and were initiated by adding Hyp into reaction mixtures, which were then incubated for 21 hr at 22°C. Upon removal from the anaerobic chamber, reactions were quenched with a 2 × volume of methanol, and precipitated protein was removed by centrifugation (15,200 g, 10 min). LC-MS/MS analysis of Pro, Hyp, and deuterated products were performed on an Agilent 6410 Triple Quadrupole LC-MS instrument (Agilent Technologies) using a Luna SCX column (5 μm, 100 Å, 50 × 2.0 mm, Phenomenex). Precursor and product ions listed in Figure 6—source data 1 were monitored.
3
Quantification of Proline and Hydroxyproline
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All assays were prepared as previously described Levin et al. (2017) (link). Briefly, assays contained 20 mM Tris-HCl pH 7.5, 100 mM KCl, 0.8 mM NADH, 3 µM P5CR, 0.2 mM Hyp, and 0.3 µM HypD. HypD was first activated under conditions described for EPR spectroscopic assays. All assays were carried out in triplicate and were initiated by adding Hyp into reaction mixtures, which were then incubated for 21 hr at 22°C. Upon removal from the anaerobic chamber, reactions were quenched with a 2 × volume of methanol and protein precipitates were removed by centrifugation (15,200 g, 10 min). Supernatants were further diluted 30-fold with water for Pro detection and 7.5-fold for Hyp detection by LC-MS/MS using previously published methods (Levin et al., 2017 (link)). Briefly, LC-MS/MS analysis of Pro and Hyp were performed on an Agilent 6410 Triple Quadrupole LC-MS instrument (Agilent Technologies) using a Luna SCX column (5 μm, 100 Å, 50 × 2.0 mm, Phenomenex). Precursor and product ions of m/z 116.1 and m/z 70.1 were monitored for proline detection whereas m/z 132.1 and m/z 86.1 were monitored for hydroxyproline. Amino acid standards were dissolved in water and diluted to a range of concentrations to generate standard curves used to quantify Pro and Hyp in samples. Source data can be found in Figure 5—source data 1 .
4
Fractionation of iTRAQ-labeled Peptides
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For SCX chromatography, iTRAQ-labeled peptides were reconstituted with Buffer A (25% ACN, 10 mM KH2PO4, pH 3.0) to a final ten-fold volume and were centrifuged at 15, 000 rpm for 10 min. The supernatant was collected and loaded onto a Phenomenex Luna SCX column (100 A). The peptides were eluted at a flow rate of 1 ml/min using Buffer B (25% ACN, 2 M KCl, 10 mM KH2PO4, pH 3.0) as follows: a gradient of 0%–5% for 1 min, 5%–30% for 20 min, and 30%–50% for 5 min; maintained at 50% for 5 min; and then maintained at 50%–100% for 5 min. The system was maintained in 100% Buffer B for 10 min before equilibrating with Buffer A for approximately 10 min until 20 min before the next injection. The elution was monitored by measuring the absorbance at 214 nm, and fractions were collected every 1 min. The eluted peptides were finally combined into 12 pooled samples and desalted on a Strata XC18 column. The fractions were dried using low-temperature centrifugation and resuspended in 1% (v/v) formic acid.
5
Venom Peptide Fractionation by HPLC
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Two different HPLC approaches were applied, including strong cation exchange (SCX) and high-pH reverse phase (Hp-RP) chromatography. In the Hp-RP procedure, 100 μg of venom peptides was separated by a Gemini Hp-RP column (4.6 × 250 mm, 5 μm, 110 Å; Phenomenex Inc.). Fractionation was performed using a linear gradient of 0%–40% of buffer A (80% ACN, 20 mM NH4FA, pH 10) at a flow rate of 1 mL/min for 40 min, 40%–90% of buffer A for 2 min, and 90% of buffer A for 3 min. In the SCX HPLC procedure, 100 μg of venom peptides was separated by a Luna SCX column (4.6 × 250 mm, 5 μm, 110 Å; Phenomenex Inc.). Fractionation was performed using a linear gradient of 0%–40% of buffer B (25% ACN, 1 M KCl, 10 mM KH2PO4, pH 3.0) at a flow rate of 1 mL/min for 40 min, 40%–90% of buffer B for 2 min, and 90% of buffer B for 3 min. All the HPLC procedures were manipulated in a 20AD HPLC system (Shimadzu, Kyoto, Japan). Absorbance was monitored at 214 nm, and the fractions were collected along the gradient for lyophilization.
6
Cross-Linked Peptide Enrichment Protocol
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