Strata x 33 μm polymeric reversed phase column

Labeled samples were pooled and subjected to the SCX fractionation column connected with an HPLC system (LC-20ab, Shimadzu, Kyoto, Japan). Peptides were eluted using buffer-1 (25 mM NaH₂PO₄ in 25% ACN, pH 2.7) and a gradient of buffer-2 (25 mM NaH₂PO₄, 1 M KCl in 25% ACN, pH 2.7). The fractionating procedure was as follows: 100% buffer A for 10 min, 5%–35% buffer B for 20 min, 35%–80% buffer-2 for 1 min. Flow rate was kept at 1 mL/min. Fractions were desalted using a Strata X 33-μm Polymeric Reversed Phase column (Phenomenex, Torrance, CA, USA) and vacuum-dried.
Peptide fractions were analyzed using Nano HPLC (LC-20AD Shimadzu, Kyoto, Japan) and a 10-cm eluting C18 column (Shimadzu, Kyoto, Japan). A Triple TOF 5600 instrument (AB SCIEX, Concord, ON, Canada), fitted with Nanospray III (AB SCIEX) and a pulled quartz-tip emitter (New Objectives, Woburn, MA, USA), was used for mass spectrometry [49 (link)]. This procedure was carried out by Guangzhou Gene denovo Biotechnology Co., Ltd. (Guangzhou, China).

The protein samples (50 μg each) were acetone-washed, reduced, alkylated and trypsin-digested according to the iTraq protocol (Sciex, Framingham, MA, USA). All labelled samples were combined to make a pooled sample. The peptides were desalted on a Strata-X 33-μm polymeric reversed phase column (Phenomenex, Torrance, CA, USA) and dissolved in a buffer containing 2% acetonitrile and 0.1% formic acid before separation by high pH on an Agilent 1100 high-performance liquid chromatography system (HPLC) using a Zorbax C18 column (2.1 × 150 mm, Agilent Technologies, Palo Alto, CA, USA). The peptides were eluted with a linear gradient of 20 mM ammonium formate, 2% acetonitrile to 20 mM ammonium formate, and 90% acetonitrile at 0.2 ml/min. The 95 fractions were concatenated into 12 fractions and dried. Each fraction was analyzed by electrospray ionization mass spectrometry using the Shimadzu Prominence nano HPLC system (Shimadzu Corporation, Kyoto, Japan) coupled to a 5600 Triple time-of-flight mass spectrometer (Sciex). The peptides were loaded onto a Zorbax 300SB-C18 3.5-μm column (Agilent Technologies) and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). Two types of samples, + agmatine and control, were used, and 2 × 4 (independent experimental samples) = 8 samples were analyzed simultaneously.