10 plex tmt kit

Isolated microglia in lysis buffer were sonicated for 3 cycles consisting of 5 s of active sonication at 30% amplitude followed by 15 s on ice. Protein concentration was determined by bicinchoninic acid (BCA) assay (Pierce, Cat. No. 23225). Protein digestion was performed as previously described [28 (link)]. Briefly, 6 μg of protein for each sample was reduced with 1 mM dithiothreitol (DTT) at room temperature for 30 min and alkylated by 5 mM iodoacetamide (IAA) in the dark for 30 min. Samples were then diluted (8-fold) with 50 mM triethylammonium bicarbonate (TEAB), digested overnight with Lysyl endopeptidase (Wako, Cat. No. 127–06621) at 1:100 (w/w). The peptide solutions were acidified to a final concentration of 1% formic acid (FA) and 0.1% triflouroacetic acid (TFA), desalted with a C₁₈ Sep-Pak column (Waters, Cat. No. WAT054945) and dried down in a vacuum centrifuge (SpeedVac Vacuum Concentrator). TMT labeling of peptides was performed according to manufacturer’s instructions and as previously described [28 (link)]. One batch of 10-plex TMT kit (Thermo Fisher, Cat. No. 90110) was used to label all 10 samples (Fig. 1b). All 10 channels were then combined and dried in a vacuum centrifuge.

Proteins from CM were isolated by transferring the supernatant to five equivalents of ice-cold acetone. Proteins were reduced using 5 mM dithiothreitol (DTT), followed by 15 mM iodoacetamide blocking before trypsination overnight at 37 °C at a protein:trypsin (Promega, Madison, WI, USA) ratio of 50:1 w/w. 10 µg of the tryptic digest was labeled with a 10-plex TMT-kit (Thermo Scientific), resuspended in anhydrous ethanol, and a 40 µg sample was labeled according to the scheme in Supplementary Table 1. Labeled samples were pooled in equal ratios, dried in a vacuum centrifuge, re-dissolved in 50 µl trifluoroacetic acid solution (0.1%), purified, loaded on a reverse phase microcolumn (equal w/w amounts of Poros R2 and Oligo R3 material) and fractionated by high pH liquid chromatography as described^{75 (link)}.
The Fractions were analyzed by RP‐nanoLC‐MS/MS on an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) equipped with a nano HPLC interface (Dionex UltiMate 3000 nano HPLC) as described^{75 (link)}. Raw data files were quantified using Proteome Discoverer version 2.4 (Thermo Scientific) as previously described using human and bovine database searches^{76 (link)}.

Aortic tissues were lysed in lysis buffer at 70 Hz for 120 s. The lysed tissue samples were centrifuged, and the supernatant was collected. The protein concentration was quantified using the Bradford method [24 (link)]. After quantification, the protein extracted from each sample was digested with 0.5 μg·μL⁻¹ trypsin solution (Thermo Fisher Scientific, NJ, USA). Then, TMT labelling was performed with a 10-plex TMT kit (Thermo Fisher Scientific, NJ, USA). Peptides in each group were labelled with different TMT labels: three biological repeats of the wild-type mice group were labelled TMT-127N, TMT-127C, and TMT-128N; three biological repeats of the ApoE^−/− group were labelled TMT-128C, TMT-129N, and TMT-129C; and three biological repeats of the berberine 156 mg·kg⁻¹ group were labelled TMT-130N, TMT-130C, and TMT-131. All the labelled samples were mixed, vacuum dried, and stored at -20°C for further analysis.

Tandem Mass Tag (TMT) was used to label the digested protein peptides according to the manufacturer’s protocol for the 10-plex TMT kit (Thermo Fisher Scientific, Waltham, MA, USA). Of the 10 labels included in each kit, 6 were used per biological pool from each treatment. Labelled peptides were then purified and separated into 10 fractions by strong cation exchange (SCX) across an increasing salt concentration. The eluted peptide fractions were purified and extracted once again before being lyophilized for direct analysis by liquid chromatography-tandem mass spectrometry (LC–MS/MS).
Fractionated samples were resuspended in 100 μL of 50 mM ammonium bicarbonate, and 10 μL of each of the 10 fractions was loaded onto a 50-cm EASY-spray column (Thermo Fisher Scientific). Quantitative analysis was performed using an Orbitrap Velos-Pro mass spectrometer (Thermo Fisher Scientific) in positive ion mode. The peptides were separated by gradient elution from 5–80% 0.1% trifluoroacetic acid in acetonitrile (5–40% from 0 to 100 min, 40–80% from 100 to 110 min) at a flow rate of 300 nL/min. Mass spectra (m/z) ranging from 400 to 1600 Da were acquired at a resolution of 60,000, and the 10 most intense ions were subjected to MS/MS by HCD fragmentation with 35% collision energy.