Tmt mass tagging kit

The 6-plex tandem mass tag (TMT) labeling was carried out according to Thermo Scientific’s TMT Mass Tagging Kits protocol. Each of the TMT 6-plex label reagents was reconstituted in 45 µL of acetonitrile (ACN), and the digested peptides from each sample were incubated with a specific tag (tags 128N, 129N, 129C used for 3 MG.MSTN^+/− samples and tags 130N, 130C, 131 used for 3 MG.WT samples, respectively) for 1 h in room temperature (Figure 12). And then the samples were pooled and dry-out to cation exchange chromatography. The hpRP chromatography was carried out as described previously (Yang et al., 2011 (link)). Forty-eight fractions were obtained at 1 min intervals and pooled into 6 fractions based on UV absorbance at 214 nm and with a multiple fraction concatenation strategy (Wang et al., 2011 (link)). The trypsin peptides labeled in each component are used for global proteomic analysis of nanoLC-MS/MS analysis.

TMT labeling was performed with TMT Mass Tagging Kits (Thermo Fisher Scientific, San Jose, CA, USA). Tags 126, 127, 130, and 131 were used for DMSO, nifedipine, nortriptyline, and metformin samples, respectively. The labeling efficiency of TMT was verified with an EASY-nLC 1,000 system coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). After labeling assessment, the TMT-tagged peptides from each sample were pooled and desalted with Sep-Pak C18 cartridges (Waters, Milford, MA, USA) before fractionation.

The peptides were desalted using the Strata X C18 solid-phase extraction column (Phenomenex, Torrance, CA, USA) after digestion with trypsin, then redissolved in 0.5 M triethylammonium bicarbonate. TMT labeling was performed using TMT mass tagging kits (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions: the thawed labeling reagents were dissolved in acetonitrile, and incubated for 2 h at 25 °C after mixing with the peptides. Finally, the peptide segment mixture was desalted and freeze-dried using vacuum centrifugation.
Subsequently, the peptide segment mixture obtained after enzymatic hydrolysis and desalination was separated using high-pH reversed-phase high-performance liquid chromatography on the Strata X C18 SPE column (Phenomenex, Torrance, CA, USA). The gradient was comprised of an increase from 6% to 23% of solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23% to 35% in 8 min, and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system.

Lysis buffer was prepared as follows: 1 mL lysis buffer (40 mM Tris-HCl and 8 M urea) containing 1 μL protease inhibitor (Thermo Fisher Scientific, Shanghai, China), 5 μL phosphatase inhibitor, and 10 μL phenylmethylsulfonyl fluoride (PMSF) (Thermo Fisher Scientific, Shanghai, China). Once prepared, the buffer was placed on ice for a few minutes before use. Frozen hepatopancreas (100 μg) was lysed with ice-cold lysis buffer. The mixture was centrifuged at 12,000× g at 4 °C for 20 min after sonicated for 5 min. The supernatant was put in a pre-cooled tube, placed on ice for 10 min, and was violently shaken 2–3 times. The homogenate was then centrifuged at 15,000× g for 10 min at 4 °C, and the total protein was placed in a new pre-cooled tube. Finally, the protein concentration was tested using the BCA Protein Assay Kit (Beyotime, Shanghai, China). The quality of extracted proteins was tested by 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Then 100 μg protein per sample were digested using trypsin (Promega, Madison, WI, USA) (trypsin ratio = 50:1) at 37 °C for 12 h and TMT reagents from TMT^® Mass Tagging Kits (Thermo Fisher Scientific, Karlsruhe, Germany) were used for peptide labeling, following the manufacturer’s instructions.

All sample peptides were labeled as previously described [21 (link)] using TMT Mass Tagging kits (Thermo Scientific, MA, USA). Briefly, peptides were resuspended in 100 μL of triethylammonium bicarbonate (TEAB). A pooled sample for normalization between runs was prepared by combining 6.25 μg of peptide from each sample. TMT labeling was accomplished on 25 μg of peptides by adding 40 μL of TMT label previously resuspended in acetonitrile. Samples were randomly distributed between 10-plex label sets with two pooled samples per set and submitted to UC Davis Proteomics Core Facility for LC-MS/MS analysis.

The digestion was quenched by the addition of 10% formic acid water, and peptides were immediately cleaned and concentrated using microspin columns (NEST group). The peptide elute was dried and reconstituted in 100 mM HEPES buffer, pH 8.5. The TMT labels were reconstituted in absolute ethanol and added to the peptide pool following instructions provided with the TMT Mass Tagging Kits and Reagents from Thermo Scientific (Waltham, MA). Protein samples extracted from LDs isolated from granulosa cells loaded with HDL or loaded with fatty acids were labeled with TMT-128 and TMT-129, respectively.