Empore spe cartridges c18

Peptides were fractionated by SCX chromatography using an AKTA Purifier system (GE Healthcare). The dried peptide mixture was reconstituted and acidified with 2 ml of buffer A (10 mM KH₂PO₄ in 25% of acetonitrile (ACN), pH 2.7) and loaded onto a Poly SULFOETHYL 4.6 × 100 mm column (5 µm, 200 Å; PolyLC Inc, Maryland, USA). The peptides were eluted at a flow rate of 1 ml/min using a gradient of 0–10% buffer B (500 mM KCl, 10 mM KH₂PO₄ in 25% of ACN, pH 2.7) for 2 min, 10–20% buffer B for 25 min, 20–45% buffer B for 5 min, and 50%–100% buffer B for 5 min. The eluents were monitored by absorbance at 214 nm and fractions were collected every 1 min. The collected fractions (approximately 30) were combined into 25 pools and desalted on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml; Sigma Aldrich, St. Louis, MO, USA). Each fraction was concentrated by vacuum centrifugation and reconstituted in 40 µl of 0.1% (v/v) trifluoroacetic acid. All samples were stored at −80 °C until LC-MS/MS analysis could be conducted.

Approximately 250 μg of protein from each sample was digested using the filter-aided sample preparation (FASP) method as previously described [19 (link)]. Put it in simple, a protein sample was suspended in UA buffer (8 M urea, 150 mM Tris-HCl, pH 8.0) to removed detergent, DTT and other low-molecular-weight components by repeated ultrafiltration (Microcon units, 10 kD). After ultrafiltration, to block reduced the cysteine residues, the 100 μL of iodoacetamide (100 mM IAA in UA buffer) was added and incubated for 30 min in the dark. Next, the filter was washed with 100 μL of UA buffer at 14,000 g for 10 min and twice with 0.025 M (100 μL) ammonium bicarbonate. Then, the protein samples were added to 100 μL of trypsin stock solution (8 μg of trypsin in 100 μL of NH₄HCO₃) for digested proteins by gentle overtaxing for 20 s and incubated at 37°C for 16 h. The digested peptides in each sample were desalted on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 mL; Sigma) and then concentrated by centrifugation at 14,000 g for 10 min and were reconstituted in 50 μL of 0.1% (v/v) trifluoroacetic acid. Based on the frequency calculation of tryptophan and tyrosine in vertebrate proteins, the peptide content was quantified by using extinction coefficient of 1.1 with 0.1% (wt/vol) solution under UV light spectral density at 280 nm.

According to our previous description (Hu et al., 2015 (link)), iTRAQ-labeled peptides were fractionated by strong cation exchange chromatography using the AKTA Purifier system (GE Healthcare). The dried peptide mixture was reconstituted and acidified with 2 ml buffer A (10 mM KH₂PO₄ in 25% ACN, pH 2.7) and loaded onto a PolySULFOETHYL 4.6 × 100 mM column (5 μm, 200 Å, PolyLC Inc, Maryland, USA). The peptides were eluted at a flow rate of 1 ml/min with a gradient of 0–10% buffer B (500 mM KCl, 10 mM KH₂PO₄ in 25% of ACN, pH 2.7) for 2 min, 10–20% buffer B for 25 min, 20–45% buffer B for 5 min, and 50–100% buffer B for 5 min. The elution was monitored by absorbance at 214 nm, and fractions were collected every 1 min. The collected fractions (about 30 fractions) were finally combined into 10 pools and desalted on C18 Cartridges [Empore™ SPE Cartridges C18 (standard density), bed inner diameter 7 mm, volume 3 ml, Sigma]. Each fraction was concentrated by vacuum centrifugation and reconstituted in 40 μl 0.1% (v/v) trifluoroacetic acid. All samples were stored at −80°C until LC-MS/MS analysis.

The resulting peptide mixture from each sample was labeled using iTRAQ reagent according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). The leaf samples collected from healthy plants at 37 WAG and 48 WAG were labeled as respective controls.
The iTRAQ-labeled peptides were fractionated by strong cation exchange (SCX) chromatography using the AKTA Purifier system (GE Healthcare, Uppsala, Sweden) by the following steps: reconstituted and acidified the labeled peptides with buffer A (10 mM KH₂PO₄ in 25% of acetonitrile, pH 3.0), and then loaded onto a PolySULFOETHYL 4.6×100 mm column (5 μm, 200 Å, PolyLC Inc, Colombia, MD, USA) and eluted at a flow rate of 1 ml min⁻¹ with a gradient of 0–8% buffer B (500 mM KCl, 10 mM KH₂PO₄ in 25% of acetonitrile, pH 3.0) for 22 min, followed by 8–52% buffer B during 22–47 min, 52–100% buffer B during 47–50 min, 100% buffer B during 50–58 min and finally buffer B was reset to 0% after 58 min. The elution was monitored by measuring the absorbance at 214 nm, and fractions were collected at every 1 min. The eluted peptides were desalted with C18 Cartridges (Empore SPE Cartridges C18, bed I.D.7 mm, volume 3 mL, Sigma) and concentrated by vacuum centrifugation.

Protein solution (30 μl) was taken and DTT was added to a 10 mM final concentration, and boiled for 5 min, and then cooled to room temperature. UA buffer (200 μl; 8 M urea, 150 mM Tris-HCl, pH 8.0) was added and centrifuged at 14 000 g for 15 min for two times. UA buffer (100 μl; 100 mM iodoacetamide in UA) was added by vortex at 600 r.p.m. for 1 min. The samples were incubated for 30 min in darkness, and centrifuged at 14 000 g for 15 min. Dissolution buffer (100 μl; AB SCIEX, Foster City, CA, USA; DS buffer) was added and centrifuged at 14 000 g for 15 min for two times. Proteins for each sample were incorporated into 30 μl SDT buffer. Then, 100 μl iodoacetamide (100 mM indole-3-acetic acid in UA buffer) was added to block reduced cysteine residues. Finally, the protein suspensions were digested with 4 μg Trypsin (Promega, Madison, WI, USA) in 40 μl DS buffer overnight at 37 °C, and the resulting peptides were collected as a filtrate.^{23 (link)} The filtrated peptides of each sample were desalted on C18 Cartridges (Empore SPE Cartridges C18, bed I.D. 7 mm, volume 3 mL, Sigma, St Louis, MO, USA), concentrated by vacuum centrifugation and reconstituted in 40 μL of 0.1% (v/v) formic acid. The peptide content was estimated by ultraviolet light spectral density at 280 nm using an extinction coefficient of 1.1 of 0.1% (g L⁻¹) solution.

The peptide mixture was constituted again and acidified with buffer A (10 mM KH₂PO₄ in 25% of ACN, pH 3.0), and loaded onto a polysulfethyl column (5 µm, 4.6 × 100 mm, 200 Å, PolyLC Inc., Columbia, MD, USA). The peptides were fractionated at a flow rate of 1 mL/min with a gradient of 0–10% buffer B (500 mM KCl, 10 mM KH₂PO₄ in 25% of ACN, pH 3.0) for 30 min, 10–60% buffer B during 30–50 min, 60–100% buffer B during 50–55 min, 100% buffer B during 55–60 min, and finally, buffer B was set to 0% after 60 min. The detector was set at 214 nm, and fractions were collected every 1 min. The collected fractions were desalted on C18 Cartridges (Empore™ SPE Cartridges C18, Sigma, St. Louis, MO, USA) and concentrated by vacuum centrifugation.

After behavioral tests, the whole brain was rapidly removed, and PFC was dissected from the brain. Liver and cecum samples were also rapidly obtained. All tissues were quick frozen in liquid nitrogen and then stored at −80 °C. Serum was immediately separated by centrifugation at 3000 rpm for 20 min at 4 °C and then stored at −80 °C. Samples from 15 FMT-treated mice with depression-like phenotype and 15 control mice were prepared for proteome fractionation. The sample size was calculated by the power analysis with a Cohen’s d effect size of 0.8. All tissues were homogenized in SDT buffer (4% SDS, 100 mM Tris-HCl, 1 mM DTT, pH 7.6) [33 (link)]. Proteins from five mice per group were pooled as a biological sample, and three biological replicates were obtained for each group. Proteins were digested with trypsin (Promega, Madison, WI, USA) in dissolution buffer overnight at 37 °C. Peptides were purified on C18 Cartridges (Empore™ SPE Cartridges C18, bed I.D. 7 mm, volume 3 ml; Sigma, Steinheim, Germany), concentrated by vacuum centrifugation, and reconstituted in 0.1% (v/v) formic acid.