Itraq reagent 4plex multiplex kit

The S180 cells were treated with 5 μM MS275 or 0.4% (v/v) DMSO as control group for 48-h incubation and the sufficient samples were submitted for iTRAQ quantitative proteomics analysis (BGI, Shenzhen, China). Protein extraction, SDS-PAGE purification, protein digestion and peptide quantification was dealt as reference [1 (link)]. The peptide samples were respectively labeled using the iTRAQ Reagent-4plex Multiplex Kit (AB SCIEX) according to the manufacturer's instructions. The labeled peptide fractionation was carried out by using Shimadzu LC-20 AB liquid phase system with 5  μm 4.6 × 250 mm Gemini C18 column and followed by HPLC (Thermo UltiMate 3000 UHPLC). The nanoliter liquid phase separation end was directly connected to the mass spectrometer with a tandem mass spectrometer Q-Exactive HF X (Thermo Fisher Scientific, San Jose, CA). The MS/MS data were searched against the Mascot database (uniprot-human 20151227.fasta) for peptide identification and quantification. The searching result of peptides was filtered by FDR p value with a cut off of 0.05. Based on statistical dispersion of the dataset, ratio of > 1.5 or < 0.667 was used as a strict significance cutoff to acquire a short list of the differentially distributed proteins as indicated in the data legends.

Filter aided proteome preparation (FASP) was performed as previously described for proteins digestion [39 (link)]. The finally filtrates were collected and the peptides were quantified at 280 nm. According to the manufacturer’s instructions, 100 µg samples from each group were labeled by iTRAQ Reagent-4plex multiplex kit (AB SCIEX, Foster City, CA, USA). Proteins from RRDCD group and RRD group were labeled with reagent 114 and reagent 116 respectively. Blank control group were labeled with reagent 117.

Eighty micrograms of the peptides were labeled using the iTRAQ Reagent-4Plex Multiplex Kit (AB SCIEX) according to the manufacturer’s protocol. The four treatments, CK, ALA, drought, and D + A, were labeled with iTRAQ tags 114, 115, 116, and 117, respectively. The separation of the iTRAQ-labeled peptides by SCX was performed on an AKTA Purifier 100 (GE Healthcare). The iTRAQ-labeled peptides that were prepared as above were mixed, and 10× volume buffer A (10 mM KH₂PO4, 25% v/v ACN, pH was adjusted to 3.0 with phosphoric acid) was added prior to loading the samples onto a polysulfoethyl 4.6 × 100 mm SCX 200 Å column containing 5 μm particles (PolyLC Inc., Maryland, U.S.A.). The peptides were eluted at a flow rate of 1 ml/min with a gradient of 100% buffer A for 25 and 25.01 min, 90% buffer A and 10% buffer B (10 mM KH₂PO₄ pH 3.0, 500 mM KCl, 25% CAN) for 32 and 32.01 min, 80% buffer A and 20% buffer B for 42 and 42.01 min, 55% buffer A and 45% buffer B for 47 and 47.01 min, 100% buffer B for 52 and 60 min, and 100% buffer A for 60.01 and 75 min. Then, in total, 33 fractions were freeze-dried and desalinized by a C₁₈ cartridge prior to liquid chromatography-mass spectrometry (LC-MS) analysis.