Orbitrap fusion mass spectrometer

The digests were analyzed in triplicate on a nanoLC-MSMS system composed of a Thermo nLC EASY1000 interfaced with a Thermo Fisher Scientific Fusion Orbitrap mass spectrometer. There was a range of peptide concentrations in these digests; trypsin digests were the most concentrated, and Asp-N digests were the least concentrated. Trypsin (2 μL), chymotrypsin (3 μL), and AspN (4 μL) digest samples were loaded on a 100 μm id × 20 mm × 5 μm 200 Å AQC18 trap column. Trapped peptides were eluted and separated on a 75 μm id × 18 cm × 5 μm 100 Å AQC18 analytical column using a 120 min nanoUPLC run. Reversed phase chromatography was performed using 0.1% formic acid in water and 0.1% formic acid in acetonitrile as mobile phases A and B, respectively. Mass spectrometric data were collected using a 3 sec top-speed data-dependent acquisition (DDA) method in which full MS scans were collected on an Orbitrap detector at a resolving power of 120,000 over a m/z 350–1500 range. Peptide precursors were isolated for collision induced dissociation (CID), and MSMS spectra from the ion trap were collected using a rapid scan rate.

Protein bands were excised manually from Coomassie- or silver-stained gels and in-gel digested using a MassPREP robotic protein-handling system (Waters, Millford, MA, USA). Gel pieces were destained twice with 100 μl 50 mM ammonium bicarbonate (Ambic) containing 50% acetonitrile at 40°C for 10 min. Proteins were then reduced with 10 mM DTT in 100 mM Ambic for 30 min at 40°C and alkylated with 55 mM iodoacetamide in 100 mM Ambic for 20 min at 40°C followed by digestion with 0.3 μg trypsin (sequence grade, Promega, Madison, WI) in 50 mM Ambic for 5 h at 40°C. The tryptic peptides were extracted with 1% formic acid in 2% acetonitrile, followed by 50% acetonitrile twice. The liquid was evaporated to dryness and the peptides were separated on an EASY-spray column connected to an EASY-nLC 1000 system (Thermo Scientific). The peptides were eluted in a 60 min gradient (from 5–26% of buffer B (2% acetonitrile, 0.1% formic acid) in 55 min and up to 95% of buffer B in 5 min) at a flow rate of 300 nL/min and analysed on a Fusion Orbitrap mass spectrometer (Thermo Scientific). The spectra were analysed using the Mascot search engine v.2.5.1 (Matrix Science Ltd., UK).

Detailed procedures were described in our previous publications [13 (link)–15 (link)]. Briefly, 100 µg of each protein sample was prepared for trypsin digestion followed by alkylation with iodoacetamide. After chloroform–methanol precipitation, each protein pellet was digested with 1 µg trypsin overnight at 37 °C. Tryptic peptides were labeled using one of the three tandem mass tags (TMT) 6-plex reagent sets (Thermo Scientific Pierce). An equal amount of each TMT-labeled sample was combined in a single tube with SepPak purified (Waters, Ireland) using acidic reverse-phase conditions. The fractionated, labeled peptide mixtures were run on a Dionex U3000 nano-flow system (Thermo Fisher Fusion Orbitrap mass spectrometer). Chromatography was conducted in a “trap-and-load” format using an EASY-Spray source. The entire run had a flow rate of 0.3 µL/min and electrospray was achieved at 1.8 kV. The 3 runs of each age group were searched using the SEQUEST HT node of Proteome Discoverer 2.4 (Thermo Scientific). The Protein FASTA database was the Mus musculus, SwissProt tax ID = 10′090, version 2017–10-25 containing 25,097 sequences. Using a false discovery rate (FDR) of < 1%, only one unique high-scoring peptide of an identified protein was needed for addition in our results. Proteome Discoverer was also used to determine the quantitative differences between biological groups.