iTRAQ labelled peptides were then fractionated by cation exchange chromatography using high performance liquid chromatography (Waters, Inc, Milford, MA) with a Zorbax 300 SCX column (5 μm; 2.1 mm × 150 mm, Agilent, USA). Lyophilized peptides were reconstituted in 2 ml (1 ml for 4-plex) loading buffer (buffer A consisting of 10 mM KH2PO4 in 75:25 water: acetonitrile, pH 2.9). The flow rate was kept at 0.4 ml/min for 17 minutes for loading after which it was increased to 0.8 ml/min. The following gradient program was employed: 0% Buffer B (10 mM KH2PO4, 1 M KCl in 75:25 water: acetonitrile (pH 2.7) for 17 min, 0% to 50% Buffer B in 39 min, 50% to 100% Buffer B in 5 min, 100% Buffer B for 10 min, 100% to 0% Buffer B in 8 min, and 0% Buffer B for 10 min (Supplementary Fig. 1 ). Eluting peptides were monitored at 214 nm and 20 fractions were collected using a fraction collector (FC 203B, Gilson). These fractions were then lyophilized by centrifugal evaporation (eppendorf).
Zorbax 300 scx column
Manufactured by Agilent Technologies
Sourced in United States
The Zorbax 300-SCX column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of ionic compounds. It utilizes strong cation exchange (SCX) technology to facilitate the separation of positively charged analytes. The column is constructed with a silica-based stationary phase and is compatible with a variety of HPLC systems and mobile phase conditions.
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4 protocols using zorbax 300 scx column
1
iTRAQ Peptide Fractionation by Cation Exchange
2
Fractionation of Labeled Peptides by SCX
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The labeled samples from three experiments (E1, E2 and E3) were fractionated by SCX liquid chromatography using a Waters Alliance HPLC system (Waters, Milford, MA, USA) at a flow rate of 0.25 mL/min. About 150 μL of the labeled peptide samples was loaded onto an SCX guard column (4.6 mm i.d. × 12.5 mm length, Agilent Technologies, Wilmington, DE, USA), and then separated with a Zorbax 300-SCX column (2.1 mm i.d. × 50 mm length, 5 μm particles, Agilent Technologies). The mobile phase gradient was generated using buffer A (10 mM KH2PO4, 20% ACN, pH 2.85) and buffer B (1 M KCl in A, pH 2.85). The samples in 0.1% FA were loaded, followed by 10 min washing with 100% A to remove excess iTRAQ reagent. Then, the peptides were separated by a 25 min linear gradient from 100% A to 100% B. Finally, the column was washed by 100% B for 5 min, followed by column equilibration with 100% A. Fractions were collected from 12 min to 42 min as follows. Eluate from 12 min to 18 min was collected to one fraction and from 36 min to 42 min as one fraction. From 18 min to 36 min, the eluate was collected as 1 min/fraction. In total, 20 fractions were collected from each sample.
3
Tyrosine Hydroxylase Enzyme Kinetics
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The activity of purified TH and truncated forms was assayed at 37 °C7 (link). The purified enzymes were kept on ice and diluted with 0.5% (w/v) bovine serum albumin in 20 mM Na-Hepes pH 7, 200 mM NaCl and centrifuged at 10,000 × g for 5 min at 4 °C. The standard assay mixture contained 20 mM Na-Hepes, pH 7, 0.1 mg/ml catalase, 10 µM FAS, 50 µM L-Tyr and 0–800 µM DA. TH was added to a final concentration of 1 µg/ml (17 nM subunit) and preincubated at 37 °C for 1 min. The reaction was started by adding 200 µM BH4 and 5 mM DTT and stopped by adding one volume of 1% (v/v) acetic acid in ethanol after 5 min for TH, THNΔ35, THNΔ43, and THS40p and after 2 min for THNΔ70. Protein was removed by precipitation at −20 °C for 90 min followed by centrifugation at 20,000 × g for 14 min at 4 °C. The amount of L-Dopa in the supernatant was determined using a 1200 series high performance liquid chromatography (HPLC) system (Agilent technologies). The chromatographic separation was obtained using an Agilent Zorbax 300-SCX column with 20 mM HAc pH 3.5, 2% (v/v) propanol as mobile phase at a flow rate of 3 ml/min. The fluorescence detector was set to λex = 280 nm and λem = 314 nm.
4
SWATH Peptide Library Generation
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For SWATH peptide reference library generation, peptides from all biological samples (ca. 300 µg total) were pooled and separated using SCX chromatography-based fractionation as described previously [73 (link)]. Briefly, peptides were separated using a Zorbax 300-SCX column (5 µm, 4.6 × 50 mm) (Agilent) at 0.5 ml min−1 on an Agilent 1100 chromatography system. Fractions (250 µl) were collected in a microtitre plate and then pooled to give 12 fractions in total. C18 ZipTips (Millipore, Cat. No. ZTC18S096) were used for desalting. The SWATH reference library was generated from information-dependent acquisition (IDA) using the 12 fractions. The HRM Calibration Kit (Ki-3003) from Biognosys (Schlieren, Switzerland) was used as a retention time standard in all IDA and SWATH proteomics experiments. IDA and SWATH samples were injected twice (technical replicates) from 5 µg of peptides and from 0.5 µg of peptides, respectively.
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