Zorbax extended c18 2

Vacuum‐dried tryptic peptides were labeled using 8‐plex iTRAQ reagents (AB SCIEX, Redwood City, CA) in accordance with the manufacturer's instructions. The reaction was quenched by incubation in ddH₂O for 30 min and was concentrated and dried in a Savant DNA120 SpeedVac (Thermo Fisher Scientific), followed by the first dimensional fractionation on a high‐pH reverse chromatography column (ZORBAX Extended‐C18 2.1, Agilent Technologies) using a separation gradient with buffer B (10 mm ammonium dissolved in 90% acetonitrile, pH 10.0), which was increased linearly from 5% up to 30% over 40 min at a flow rate of 0.3 ml min⁻¹. A total of 40 fractions were collected at even intervals. For every minute, four fractions were combined, and 15 fractions were finally obtained and used in the LC‐MS analysis.

The derived peptides from the different samples were labelled using isotopomeric dimethyl labels, following a previously described method with some modifications.
³¹ (link) In brief, two C18 SepPak column were washed twice with ACN and solvent A (2% ACN and 0.1% formic acid). Each of the different samples were separately loaded into a SepPak column and washed with 2 mL of solvent A. Then, the SepPak columns were flushed five times with 1 mL of the respective labelling reagent (light or heavy). Following this, 2 mL of solvent A was used to wash the SepPak columns, and then the labelled samples were eluted and collected from the SepPak columns with 500 mL of solvent B (80% ACN and 0.1% formic acid). The labelled samples were mixed and dried in speed‐vacuum concentrator. Dried samples were subjected to the first dimensional fractionation procedure on high‐pH reverse chromatography column (Agilent, ZORBAX Extended‐C18 2.1). The mobile phase consisted of buffer A (10 mM ammonium formate dissolved in 5% ACN, pH = 10.0) and buffer B (10 mM ammonium formate dissolved in 90% ACN, pH = 10.0). The elution gradient was from 5% to 30% buffer B linearly over 40 min with a 0.3 mL min⁻¹ flow rate. A total of eight fractions were collected and prepared for the following LC–MS analysis.