Scx column

To create a comprehensive neutrophil spectral library, selected healthy donor and patient peptide samples were eluted from StageTips using 80% acetonitrile (ACN) in 0.1% formic acid, dried in Eppendorf Concentrator plus 5305, reconstituted in SCX buffer A (5 mm K2HPO4 in 10% ACN) and pooled. 100 μg of pooled peptides were separated on a Shimadzu LC-20AD HPLC system using a PolyLC PolySULFOETHYL-A SCX column (100 × 2.1 mm, 3 μm beads, 300 Å pores) with a 12 min nonlinear gradient of SCX buffer B (1 m KCl, 5 mm K2HPO4 in 10% ACN) while collecting fractions every 15 s. The fractions were then concentrated, reconstituted in 0.1% TFA and desalted using C18-StageTips. Lower complexity fractions were pooled together prior to mass spectrometric analysis.

A total of 100 μg proteins for each sample were digested with trypsin gold (Promega, Madison, WI, USA) at 40:1 mass ratio at 37 °C for 12 h. After digestion, peptides were desalted using Strata X C18 column (Phenomenex) and vacuum-dried following the manufacturer’s protocol. The iTRAQ labeling of peptide was processed using iTRAQ reagent 8-plex kit following the manufacturer’s instructions. The eight samples including S_Cd_1, S_Cd_2, S_CK_1, S_CK_2, F_Cd_1, F_Cd_2, F_CK_1 and F_CK_2 were labeled with tags 113 and 114, 119, 121, 117, 118, 115 and 116, respectively, and incubated at room temperature for 2 h. For peptide fractionation, the strong cationic exchange (SCX) chromatography was carried out with a LC-20AB HPLC Pump system (Shimadzu, Kyoto, Japan) with an Ultremex SCX column (4.6 × 250 mm) as previously described [54 (link)].

After labeling and quenching, the samples of roots and leaves were combined and lyophilized, respectively. The peptide mixture was dissolved in 4 mL strong cation exchange (SCX) buffer A (25% v/v acetonitrile, 25 mMNaH₂PO₄, pH 2.7). The peptides were fractionated on a Shimadzu LC-20ABHPLC system (Shimadzu, Kyoto, Japan) using an Ultremex SCX column (4.6 × 250 mm). Peptides were eluted at a flow rate of 1 mL/min with elution buffer B (25% v/v acetonitrile, 25 mM NaH₂PO₄, 1 M KCl, pH 2.7). The absorbance at 214 nm was monitored and 20 fractions were collected. Samples of each fraction were dried and desalted before LC-ESI MS/MS analysis [33 (link),34 (link)].