Tmt labeling kit

With the fastest growing cultures (the F4C4 group, see section “Physiological response to copper and iron deduction” in Result) set as the control group, the Fe-deduction group (i.e., F1C4), Cu-deduction group (i.e., F4C1), and Cu–Fe dual deduction group (i.e., F1C1) were set as the experimental groups. Three replicates were performed for each group. These cultures have the same culture conditions and growth status as the cultures used for growth curve measurement and were investigated using TMT quantitative proteomics to determine the effect of Cu/Fe deductions on Synechocystis 6803 protein levels. Cells in logarithmic growth phase were collected for proteomic determination after 120 h of metal-deduction.
Proteins were extracted from cyanobacteria using the SDT [4% w/v sodium dodecul sulfate (SDS), 100 mM Tris/HCl pH 7.6, 0.1 M dithiothreitol (DTT)] cleavage method (Wiśniewski et al., 2009 (link)), and total protein levels were quantified using the bicinchoninic acid assay (BCA) method. Each sample was trypsinized using the filter-aided proteome preparation (FASP) method and the peptides were quantified (OD₂₈₀) (Wiśniewski et al., 2009 (link)). The peptides (100 μg) of each sample were labeled according to the instructions of the TMT labeling kit (Thermo, United States).

Firstly, according to the method given by Tang et al. (25 (link)), the protein was extracted by lysis with SDT lysate (including 4% w/v SDS, 150 mM Tris/HCl (pH8), and 100 mM DTT). Also, 30 mg of each ovarian tissue was added to 900 μL SDT; the lysate was sonicated (80 W, 10 cycles of 10 s with 15 s intervals) and then boiled for 15 min. After centrifugation at 14,000 × g for 40 min, the final supernatant was quantified using a BCA assay (Bio-Rad, CA, United States) and detected by SDS-PAGE (Supplementary Figure S2). Protein was treated with the filter-aided sample preparation (FASP) method for enzymatic hydrolysis (30 (link)), and the filtrate was collected. The peptide fraction was qualified at 280 nm, OD280. After trypsin digestion, the peptide samples were dried by vacuum centrifugation, then 100 μg peptides of each sample were resuspended in 0.5 M TEAB. The TMT labeling kit (Thermo Fisher Scientific, Waltham, MA, United States) with the isobaric labels 126, 127, 128, and 129 was used to label the PF, PL, MF, and ML samples, respectively.

Reagents were as follows: TMT Labeling Kit (Thermo Inc.), ProteoMiner Low Abundance Protein Enrichment Kit (Bio-Rad Inc.), BCA Kit (Biyuntian Biotechnology Co., Ltd.), trypsin (Promega Inc.), Trifluoroacetic Acid (Sigma-Aldrich Inc.), formic acid (Fluka Inc.), iodoacetamide (Sigma Inc.), dithiothreitol (Sigma Inc.), urea (Sigma Inc.), triethylammonium hydrogen carbonate (Sigma Inc.), ultrapure water (Fisher Chemical Inc.), and 0.26 mm–0.28 mm monofilament nylon fishing line (2838–20A4 Beijing Xion Technology Co., Ltd.). Standard: Ligustrazine Hydrochloride (110817–201608), ferulic acid (110773–201614), and astragaloside IV (110781–201717) were purchased from China Food and Drug Control Research Institute. Rat angiotensinogen ELISA kit (ER0371) and catalase kit (ER0264) were purchased from Wuhan Fine Biotechnology Co., Ltd. Anti-catalase antibody (catalog number ab209211), anti-angiotensinogen antibody (catalog number ab213705), and Anti-beta Actin (catalog number ab8226) were purchased from Abcam company.
Instruments used were as follows: Ultrasonic Cell Breaker (Xinzhi Biotechnology Co., Ltd.), high-speed refrigerated centrifuge, Dynamica microplate reader (Bio-Rad Inc.), and Scientific Q Exactive, Scientific Q Exactive Plus, Scientific Orbitrap Fusion (Thermo Inc.). Column was Welch Vltimate XB-C18 (HS), 4.6 × 250 nm, 5 μm.

One hundred milligrams of peptide were taken from each sample and labeled according to the Thermo Fisher TMT Labeling Kit instructions. The experimental induction group and the control group were labeled as TMT-126 and TMT-127, and three replicates were set. The labeled peptides were mixed in equal amounts, and the dried peptides were fractionated using High-pH (Pierce™ High pH Reversed-Phase Peptide Fractionation Kit, Thermo Fisher Scientific, Waltham, USA). The sample collection was eventually combined into ten components. The peptides of each component were dried and reconstituted with 0.1% FA for LC-MS analysis. TMT markers and LC-MS data acquisition were determined by Shanghai Baipu Biotechnology Co., Ltd. (Shanghai, China).

After trypsin digestion, the peptides were desalted using a Strata X C18 SPE column (Phenomenex) and dried by vacuum centrifugation. The desalted peptides (100 µg) from each sample were dissolved in 0.5 moL/L TEAB solution and labeled using the TMT Labeling Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Briefly, a unit of TMT reagent was thawed and reconstituted in acetonitrile. After the polypeptide mixture was incubated at RT for 2 h, it was pooled, desalted, and dried by vacuum centrifugation.

Furthermore, 100 μg peptides of each sample were labeled according to the instructions of the TMT labeling kit (Thermo Fisher, USA). The labeled peptides from each group were pooled and purified using an AKTA Purifier 100 (GE Healthcare, USA). The sample was loaded to the column equilibrated with buffer A (10 mM KH₂PO₄, 25% ACN, pH 3.0) and B (10 mM KH₂PO₄, 500 mM KCl, 25% ACN, pH 3.0). The chromatographic column (5 μm, 200 Å, PolyLCInc, Maryland, U.S.A.) was equilibrated with liquid A, and the sample was loaded from the injector to the chromatographic column for separation at a flow rate of 1 ml/min. The liquid-phase gradient was as follows: 0–25 min, the linear gradient of liquid B was from 0 to 10%; 25–32 min, the linear gradient of liquid B was from 10 to 20%; 32–42 min, the linear gradient of liquid B was from 20 to 45%; 42–47 min, the linear gradient of B solution was from 45 to 100%; 47–52 min, 52–60 min B solution was maintained at 100%; after 60 min, B solution was reset to 0%. During the elution process, the absorbance value at 214 nm was monitored, and the elution fractions were collected every 1 min, respectively, lyophilized and desalted using a C18 Cartridge.