Tmtsixplex isobaric mass tagging kit

Samples were subsequently labeled with tandem mass tags (TMTs) for quantitative mass spectrometry (TMTsixplex Isobaric Mass Tagging Kit; Thermo Fisher Scientific). Briefly, each sample was reduced with 500 mM tris(2-carboxyethyl)phosphine at 55 °C for 1 h and then alkylated with 300 mM iodoacetamide at 37 °C in the dark for 30 minutes. The samples were desalted using a 10,000 Da molecular weight cutoff membrane filter and dissolved in 100 mM triethylammonium bicarbonate (TEAB) buffer to a final concentration of 1 μg/μl. Sequencing-grade trypsin (Promega, Madison, WI, USA) was added at 1:100 (wt/wt) to the proteins in TEAB buffer and incubated overnight at 37 °C. Three samples each from the CR and non-CR groups were individually labeled using TMT-126, TMT-128, and TMT-130 (CR group) and TMT-127, TMT-129, and TMT-131 (non-CR group) following the manufacturer’s instructions. Aqueous hydroxylamine solution (5% wt/vol) was added to quench the reaction. The six samples were then combined, dried by speed-vacuum method, and then dissolved in 50 μl of water containing 0.1% formic acid for LC-MS/MS analysis.

A 100 μg proteins from each sample were precipitated in 20% trichloroacetic acid (TCA) at 4°C. Protein pellets were washed with ice-cold acetone and re-suspended in 25 μl TEAB (100 mM) and 25 μl 2,2,2-trifluoroethanol (TFE). Proteins were reduced with 1 μl of tris(2-carboxyethyl)phosphine (TCEP, 500 mM), alkylated with iodoacetamide (IAA, 30 mM), and digested with 2.5 μg/sample of trypsin/lysC (Promega, Madison, WI, United States) overnight at 37°C. The digested peptides were quantified using the Pierce Quantitative Colorimetric Peptide Assay (Thermo Scientific, Waltham, MA, United States). 40 μg of peptides from each specific sample was labeled with the Thermo Scientific TMTsixplex Isobaric Mass Tagging Kit (JSC-E1 (ground 1) with TMT⁶-128, JSC-E2 (ground 2) with TMT⁶-130, JSC-S1 (ISS 1) with TMT⁶-129, JSC-S2 (ISS 2) with TMT⁶-131) according to the manufacturer’s protocol. All labeled-peptide mixtures were combined into a single tube, mixed, and fractionated using the Thermo Scientific Pierce High pH Reversed-Phase Peptide Fractionation Kit. While this kit usually uses eight fractions with step elution of up to 50% acetonitrile, ninth fraction was added eluting at 100% acetonitrile. Nine fractionated samples were dried using a SpeedVac concentrator and re-suspended in 1% (v/v) formic acid prior to liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis.

After trypsin digestion, the peptide was desalted by Strata X C18 SPE column (Phenomenex, Torrance, CA, USA) and vacuum-dried. The peptide was reconstituted in 1 M TEAB and processed according to the protocol of the manufacturer for a TMTsixplex Isobaric Mass Tagging kit (ThermoFisher Scientific, Massachusetts, USA). Briefly, one unit of TMT reagent (defined as the amount of reagent required to label 100 μg of protein) was thawed and reconstituted in 24 μl acetonitrile (ACN). The peptide mixtures were then incubated for 2 h at room temperature and were pooled, desalted, and dried by vacuum centrifugation.

The resulting tryptic peptides were subsequently labeled using TMTsixplex Isobaric Mass Tagging kit (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions as follows: 126, WT-R1; 127, WT-R2; 128, WT-R3; 129, KO-R1; 130, KO-R2; and 131, KO-R3. After labeling, the samples were pooled, evaporated to dryness, and stored at −20°C until the LC-MS analysis. Three biological replicates of each condition were analyzed.

For digestion, 40 µg of protein from each condition were precipitated by methanol-chloroform method. Protein pellets were resuspended and denatured, as previously described by Méndez et al.^{81 (link)}. The resulting peptides were subsequently labeled using TMT-six-plex Isobaric Mass Tagging Kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions as follows: 126: C-1; 127: BFA-1; 128: C-2; 129: BFA-2; 130: C-3; 131: BFA-3. Three biological replicates of each condition were analyzed in this study. After labeling, the samples were pooled, evaporated to dryness and stored at − 20 °C until the LC–MS analysis.

The resultant peptide mixture from desalted proteins tryptic digest (60 µg) was labeled using chemicals from the TMT sixplex Isobaric Mass Tagging Kit (Thermo Fisher Scientific, MA, USA) as described by the manufacturer. Briefly, peptides were dissolved in 50 μL of 100 mM triethylammonium bicarbonate (TEAB), adjusted to pH 8. For labeling, each TMT reagent was dissolved in 41 μL of ACN and added to the respective peptide mixture and then incubated at room temperature for 1 h. Labeling was stopped by the addition of 8 μL 5% hidroxilamine. Whole supernatants were dried down and the four samples were mixed to obtain the “4plex-labeled mixture”. The mixture was analyzed by RP-LC-MS/MS to check the efficiency of the labeling.

The resultant peptide mixture (50 μg) was labelled using chemicals from the TMT sixplex Isobaric Mass Tagging Kit (Thermo Fisher Scientific, MA, USA) essentially as described by the manufacturer. Briefly, peptides were dissolved in 50 μl of 100 mM triethylammonium bicarbonate (TEAB), adjusted to pH 8. For labelling, each TMT reagent was dissolved in 41 μl of acetonitrile and added to the respective peptide mixture and then incubated at room temperature for one hour. Labelling was stopped by the addition of 8 μl 5% hydroxylamine. Whole supernatants were dried down and the six samples were mixed to obtain the “6plex‐labeled mixture” (Zhou et al., 2019). The mixture was analysed by RP‐LC‐MS/MS to check the labelling efficiency.
The sample was then fractionated using the Pierce High pH Reversed‐Phase Peptide Fractionation Kitt (Thermo Fisher Scientific, MA, USA): sample was re‐swollen in 0.1%TFA and loaded onto an equilibrated, high‐pH, reversed‐phase fractionation spin column. A step gradient of increasing acetonitrile concentrations (5%–80%) in a volatile high‐pH (Triethylamine (0.1%)) was then applied to the columns to elute bound peptides into nine different fractions collected by centrifugation, dried and stored until analysis.