Betasil c18 column

The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Thermo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm length). Briefly, peptides were first separated with a gradient of 8–32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined into six fractions and dried by vacuum centrifuging.

The tryptic peptides were fractionated by high pH reverse-phase HPLC using a Thermo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm length). Briefly, the peptides were first separated with a gradient of 8% to 32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined and dried by vacuum centrifugation.

The tryptic peptides were fractionated into fractions with the high pH reverse-phase high-performance liquid chromatography (HPLC), based on the Thermo Betasil C18 column. The peptides were separated into 60 fractions with a gradient of 8% to 32% acetonitrile (pH 9.0) (Fisher Chemical) over 60 min and then pooled into 4 and 11 fractions by combining with equal time interval for mouse hepatocyte maturation and human hepatic reprogramming, followed by the vacuum drying.
For the phosphopeptide enrichment, the loading buffer (50% acetonitrile & 6% TFA (Sigma-Aldrich)) with Ti⁴⁺-immobilized metal ion affinity chromatography (Ti⁴⁺-IMAC) microspheres was used to incubate the peptide mixtures for 1 h at room temperature (30 rpm). Next, the IMAC microspheres with enriched phosphopeptides were collected after centrifugation. Then, 50% acetonitrile with 6% TFA and 30% acetonitrile with 0.1% TFA were used to wash the microsphere for 10 min at room temperature (30 rpm) sequentially, to remove the nonspecifically adsorbed peptides. From the elution of phosphopeptides with microspheres by adding elution buffer containing 10% NH₄OH for 10 min at room temperature (30 rpm), the supernatant containing phosphopeptides was collected and lyophilized for further analysis.

The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Thermo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm length).

Peptides were fractionated by high pH reverse-phase HPLC using Thermo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm length). Briefly, peptides were first separated with a gradient of 8% to 32% acetonitrile (pH 9.0; Fisher Chemical, USA) over 60 min into 60 fractions and then were combined into 6 fractions. To enrich labeled peptides, tryptic peptides dissolved in IP buffer (150 mM NaCl, 250 mM Tris-HCl, pH 7.5) were incubated with pre-washed anti-TMT antibody beads (Thermo, USA) at 4°C overnight with gentle shaking. Peptides were enriched using anti-TMT antibody and four different labels were used for the labeling of R-SH (TMT-126 and TMT-127) and R-SOH (TMT-128 and TMT-129) in control and freezing treated samples, respectively. Then the beads were washed for four times with IP buffer and twice with ddH₂O. The bound peptides were eluted from the beads with 0.1% trifluoroacetic acid (TFA). Finally, the eluted fractions were desalted and vacuum-dried for LC-MS/MS Analysis.

The peptides were separated into fractions using high pH reverse-phase HPLC. Thermo Betasil C18 column (5 μm particles, 250 mm, and 10 mm i.d.) was used to perform the HPLC fractionation under a gradient of 8–32% acetonitrile (pH 9.0) within 60 min. Sixty fractions were collected and then combined to 10 fractions. After drying in a vacuum centrifuge, the fractions were dissolved in 0.1% formic acid for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.