Proteomics procedures are scheduled in Supplementary Fig. S3 and fully described in Supplementary Information . The peptide pools were isobarically labelled (iTRAQ, Sigma-Aldrich), equally mixed and the peptide mixture was desalted onto Oasis HLB C18 cartridges (Waters). One-fifth of the tagged peptides were dried-down, and the remaining four-fifths were separated into 8 fractions using MCX Oasis cartridges (Waters). The peptide fractions were desalted using MicroSpin Colums C18 (The Next Group) and vacuum-dried.
Mcx oasis cartridges
Manufactured by Waters Corporation
MCX Oasis cartridges are solid-phase extraction (SPE) devices designed for sample preparation and purification. They are used to extract and concentrate target analytes from complex sample matrices prior to instrumental analysis.
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Lab products found in correlation
2 protocols using mcx oasis cartridges
1
Isobaric Peptide Fractionation and Labeling
2
iTRAQ Labeling and Peptide Fractionation
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For iTRAQ (isobaric Tags for Relative and Absolute Quantification) labeling, the dried peptides were taken up in 30 μL of 0.5 M triethylammoniumbicarbonate (TEAB) buffer and labeled with the corresponding iTRAQ reagent in 70% (v/v) ethanol for 1 h at room temperature. Then, 100 μL of 0.5% (v/v) trifluoroacetic acid (TFA) was added to stop the labeling reaction. The peptide samples were mixed, vacuum concentrated and diluted in 200 μL of 1% (v/v) TFA for desalting on Oasis HLB C18 cartridges (Waters). One-fourth of the tagged peptides were directly analyzed by LC-MS, and the remaining three-fourths were subject to mixed-mode cationic exchange (MCX) fractionation. The iTRAQ-labeled peptides were suspended in 5 mM ammonium formate with 25% (v/v) acetonitrile (ACN), pH 3.0, and separated into 5 fractions using MCX Oasis cartridges (Waters). The so-obtained peptide fractions were desalted using MicroSpin Columns C18 (The Next Group), vacuum-dried and kept at 4 °C for later LC-MS analysis.
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