Tmt 10 plex reagent

The meat samples were prepared following our previous published protocols [5 (link)]. In brief, 1.5 mL of protein lysis buffer (8 M urea, 1% SDS) was used to suspend the powder of meat samples (100 mg). Then, after being incubated on ice for 30 min and sonicated for 2 min, the supernatant was collected and quantified through a BCA Protein Assay Kit (Thermo Scientific). Lastly, 9 pools (5 samples per pool and 3 pools per age stage) with the same concentration were obtained according to protein content. After being digested at 37 °C overnight with trypsin (the mass ratio of trypsin-to-protein at 1:50), the peptides were labeled with 10-plex TMT reagents (Thermo Fisher Scientific) and multiplex labeled samples were obtained.

In order to identify proteins proximal and/or physically associated with SHMT2 in living cells, the BioID method was employed [24 ]. Biotinylated proteins were affinity purified by using immobilized streptavidin, followed by trypsin digestion, MS, and bioinformatics analysis for peptide and protein identifications essentially as described previously [25 (link)].
For proteomic analysis of tandem mass tag (TMT)-labeled samples, 2% SDS lysis buffer (100 mM HEPES pH 7.3 and 2% SDS, 50 mM NaCl, 10 mM TCEP((Tris (2-Carboxyethyl) phosphine Hydrochloride), 40 mM CAA (Chloroacetamide), and complete protease inhibitor (same as in NP40 buffer)) was used to quickly lyse cells from cell culture or from tumor samples by heating at 95°C for 15 min [26 ]. Protein (50 μg) was precipitated by using methanol-chloroform and then digested with a trypsin/Lys-C mixture (1 μg, Promega Cat#V5073) [27 ]. Digested peptides (25 μg) were labeled with 10-plex TMT reagents according to the manufacturer’s instructions (ThermoFisher Scientific). Ten labelled samples as indicated were pooled and fractionated by using the Pierce high pH kit (Pierce Cat#84868). Eight fractions of each pooled TMT sample were then analyzed by using an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific) [26 ,27 ].

Cell pellets from 24 and 48 h after NC or miR-206 mimic transfection were lysed, digested and labeled as described previously.^{47 (link)} One hundred micrograms of each sample was digested with Lys-C (Wako, Richmond, VA, USA; 1:100 w/w) at room temperature for 2 h, diluted 4 × with 50 mM HEPES and further digested with trypsin (1:50 w/w; Promega, Madison, WI, USA) overnight at room temperature. Peptide samples were acidified by TFA, desalted with Sep-Pak C₁₈ cartridge (Waters, Milford, MA, USA), eluted by 60% ACN 1% formic acid and dried by speedvac. Samples were resuspended in 50 mM HEPES and labeled with 10-plex TMT reagents (Thermo Fisher Scientific). The labeled samples were equally pooled, desalted and dried by speedvac. Labeled peptides were analyzed based on an optimized LC-MS/MS platform on an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific).^{48 (link)} Acquired MS data were searched against a human protein database utilizing JUMP algorithm, a tag-based database search program, for protein identification (protein FDR<1%).^{48 (link)} Quantification was achieved by analyzing the reporter ion intensities from the MS/MS spectra.

Detergent-insoluble extract fractions were digested with modified trypsin (Promega) using the FASP protocol [59 (link)]. Equal amounts of each peptide sample were labeled using the 10-plex TMT Reagents (Thermo Fisher Scientific) and then analyzed via liquid chromatography-mass spectrometry (LC-MS/MS) [60 (link),61 (link),62 (link),63 (link)].

One hundred microgram of proteins were resuspended with tetraethylammonium bromide (Haihang Industry, Jinan, China) at a final concentration of 100 mM. The mixture was reduced with tris (2-carboxyethyl) phosphine (Sigma) at a final concentration of 10 mM at 37 °C for 60 min, and alkylated with iodoacetamide (Sigma) at a final concentration of 40 mM at room temperature for 40 min in darkness. Six fold volumes of cold acetone were added to precipitate protein at − 20 °C for 4 h. After centrifugation at 10,000×g at 4 °C for 20 min, the pellet was resuspended with 100 μL 50 mM riethylammonium bicarbonate buffer (Sigma). Trypsin was added at 1:50 trypsin-to-protein mass ratio and incubated at 37 °C overnight. Trypsin-digested peptides were labeled with 10-plex TMT reagents (Thermo) according to the manufacturer’s instructions. Briefly, one unit of TMT reagent were thawed and reconstituted in 50 μL acetonitrile (Sigma). After tagging for 2 h at room temperature, hydroxylamine (Thermo) was added to react for 15 min at room temperature. Finally all samples were pooled, desalted and vacuum-dried.