The high-pH reverse-phase liquid chromatography (RPLC) separation was performed on the 1220 Infinity LC system with a Zorbax Extended-C18 analytical column containing 1.8 µM particles (Agilent Technologies Inc., CA); flow rate was 0.2 mL/min. The mobile-phase A consisted of 10 mM ammonium formate (pH 10) and B consisted of 10 mM ammonium formate and 90% acetonitrile (pH 10). A total of 50 µg peptides were fractionated using the following linear gradient: from 0% to 2% B in 10 min, from 2% to 8% B in 5 min, from 8% to 35% B in 85 min, from 35% to 95% B in 5 min, and then held at 95% B for an additional 15 min. Peptides were detected at 215 nm and 96 fractions were collected along with the LC separation in a time-based mode from 16 to 112 min. The separated peptides in 96 wells were concatenated into 24 fractions by combining four wells into one sample, such as 1, 25, 49, and 73 as fraction one; 2, 26, 50, and 74 as fraction two; and so on. The peptides were then dried in a Speed-Vacuum and stored at −80 °C until liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as described in our Supplemental Materials and Methods section.
Zorbax extended c18 analytical column
Manufactured by Agilent Technologies
The Zorbax Extended-C18 analytical column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a silica-based stationary phase with a C18 alkyl chain modification, providing a hydrophobic surface for the retention of non-polar and moderately polar analytes. The column is suitable for use in normal-phase, reversed-phase, and ion-exchange HPLC methods.
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4 protocols using zorbax extended c18 analytical column
1
High-pH Reverse-Phase Peptide Fractionation
2
N-Glycopeptide Enrichment from Prostate Cancer Samples
3
High-pH RPLC Peptide Fractionation
4
GC-MS and HPLC Analysis of CTLE
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GC-MS analysis was performed as reported previously with slight modifications [20 (link)]. An Agilent 7890 gas chromatograph system was coupled to a quadrupole Agilent 5975C electron ionization (70 eV) mass spectrometric detector (Agilent Technologies, Palo Alto, CA, USA). The operational parameters for the GC-MS analysis are listed in Table 1 . In addition, we purified and identified two constituents (rutin and kaempferol) by using preparative column liquid chromatography (prep-LC) and preparative thin layer chromatography. The analytical conditions for prep-LC are summarized in Table 1 . Then, constituent profiling of CTLE was performed by using an Alliance 2695 HPLC system (Waters; Milford, MA, USA) coupled to a photodiode array detector at the wave length of 320 nm. An Agilent Zorbax extended C18 analytical column (150 mm × 5 mm; 5 μm) was used with a filtered and degassed mobile phase that consisted of solvents A (acetonitrile) and B (0.2% phosphoric acid). Gradient elution (from 10/90 to 100/0, v/v) was performed at flow rate of 0.8 mL/min. The column temperature and sample injection volume were 25°C and 10 μL, respectively.
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