Phosphate elimination was carried out using 5 μM phosphothreonine lyase and 25 μΜ peptide in reaction buffer (25 mM Tris, 150 mM NaCl, pH 8.0). For phospho-peptides that mimic the activation loop of ERK, peptides were treated with His6-SpvC or His6-OspF at 30 °C for 90 min. For the phospho-Akt peptide, peptide was treated with His6-OspF at 30 °C for 18 h. The phosphate elimination process was monitored by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and dehydroamino acid containing peptides were purified by RP-HPLC (Macherey-Nagel, VP 250/10 Nucleodur C18 HTec, 5 μm). For glutathionylation of the ERK peptide, 25 μM Dhb-containing peptide was dissolved in reaction buffer (25 mM Tris, 150 mM NaCl, 1 mM glutathione (GSH), 1 μM TCEP, pH 8.0) and incubated with 5 μM LanCL2-His6 at 25 °C for 18 h. The glutathionylation process was monitored by MALDI-TOF MS and the resulting peptide was purified by RP-HPLC (Macherey-Nagel, VP 250/10 Nucleodur C18 HTec, 5 μm). All peptides described in this section were purified by HPLC as described in the peptide synthesis section.
Vp 250 10 nucleodur c18 htec
Manufactured by Macherey-Nagel
The VP 250/10 Nucleodur C18 HTec is a high-performance liquid chromatography (HPLC) column designed for analytical separations. It features a C18 bonded stationary phase and is intended for use in various analytical applications.
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2 protocols using vp 250 10 nucleodur c18 htec
1
Phosphate Elimination and Glutathionylation of Peptides
2
Analytical and Preparative HPLC-PDA Protocol
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The analytical HPLC-PDA system consisted of an Agilent HP1100 series (binary pump G1312A, auto sampler G1313A, and photodiode array detector G1315B, 200-700 nm; Agilent, Waldbronn, Germany), equipped with an EC250/4 Nucleodur C18 HTec column from Macherey-Nagel (5 μm; injection volume 20 μL). Method 1: binary gradient linearly increasing in 21 min from 20 to 67% methanol in acidified water (0.1% (v/v) trifluoroacetic acid, purity > 99.9%, Roth), subsequent washing step (100% methanol) and equilibration at starting conditions. The flow rate was 1 ml min À 1 and the detection wavelengths were 211, 254, 281, 351 and 460 nm.
For preparative HPLC, a LC-20AT chromatography system (Shimadzu, Kyoto, Japan) equipped with autosampler SIL-10AP, fraction collector FRC-10A and UV/vis detector SPD-20A was used. The separation column was a VP250/10 Nucleodur C18 HTec, 5 μm (Macherey Nagel). Method 2: 2-min focusing step with 30% acidified methanol in acidified water (0.1% (v/v) HCOOH), followed by a gradient linearly increasing in 33 min to 50% methanol, washing, and re-equilibration of the column (flow rate 3.5 ml min À 1 ). The detector was operating at 351 and 500 nm and the seven most abundant peaks at 351 nm were collected. Therefore, collection was performed between 24 and 50 min for peaks with higher intensity levels than 8000 μV.
For preparative HPLC, a LC-20AT chromatography system (Shimadzu, Kyoto, Japan) equipped with autosampler SIL-10AP, fraction collector FRC-10A and UV/vis detector SPD-20A was used. The separation column was a VP250/10 Nucleodur C18 HTec, 5 μm (Macherey Nagel). Method 2: 2-min focusing step with 30% acidified methanol in acidified water (0.1% (v/v) HCOOH), followed by a gradient linearly increasing in 33 min to 50% methanol, washing, and re-equilibration of the column (flow rate 3.5 ml min À 1 ). The detector was operating at 351 and 500 nm and the seven most abundant peaks at 351 nm were collected. Therefore, collection was performed between 24 and 50 min for peaks with higher intensity levels than 8000 μV.
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