Ultremex column

The labelled samples were fractionated using a high-performance liquid chromatography (HPLC) system (LC-20AB, Shimadzu, Japan) connected to an SCX column (Ultremex column, 4.6 mm I.D. × 250 mm, Phenomenex). The retained peptides were dissolved using 4 mL of buffer A (25 mM NaH₂PO₄ in 25% ACN, pH 2.7). After the peptides flowed onto the columns, the retained peptides were eluted using Buffer A for 10 min and 5–35% Buffer B (25 mM NaH₂PO₄, 1 M KCl in 25% ACN, pH 2.7) for 20 min, and then eluted using 35–80% buffer B for 1 min. The flow rate was set at 1 mL/min. Fractions were collected in 1.5 mL microfuge tubes every minute starting at 15 min after sample injection, and a total of 10 fractions was collected. The salt was removed from fractions with a high salt content using a Strata-X 33 μm Polymeric Reversed Phase column. The eluted fractions were dried in a vacuum concentrator and then dissolved in 0.1% formic acid prior to reverse-phase nLC-tandem mass spectrometry.

SCX was conducted on a high-performance liquid chromatography (HPLC) system (LC-20AB, Shimadzu, Tokyo, Japan) with an SCX column (Ultremex column, 4.6 mm I.D. × 250 mm, Phenomenex, Torrance, CA, USA). The retained peptides were dissolved using 4 mL buffer A (25 mM NaH₂PO₄ in 25% ACN, pH 2.7), and eluted using Buffer A for 10 min, 5–35% Buffer B (25 mM NaH2PO4, 1M KCl in 25% ACN, pH2.7) for 20 min, and 35–80% buffer B for 1 min, with at flow rate set at 1 mL/min when peptides flowed into the columns. The fractions were collected every 15 min after sample injection and desalted using a Strata-X 33-μm Polymeric Reversed Phase column, then dried in a vacuum concentrator and dissolved with 0.1% formic acid prior to reverse-phase nano-liquid chromatography/tandem mass spectrometry (nLC-MS/MS).