Protocols

Proteomic analysis

Tissue homogenisation

For proteomics, tissue samples that had been frozen in liquid nitrogen and stored at –800C were homogenised in tubes with ceramic beads (Precellys ceramic kit 1.4mm beads, VWR, 432-923) using a Minilys homogeniser (5000rpm, 5 cycles, 25 seconds each) in lysis buffer (5% SDS/50mM TEAB, pH 8) containing protease and phosphatase inhibitors. Homogenised samples were centrifuged at 13,000 rpm for 10 minutes at 4⁰C and the supernatant was collected and stored at -80⁰C until further use. Protein concentration of each sample was estimated using Pierce BCA Assay kit (Thermo Fisher Scientific) with absorbance read at 562nm.

Peptides Extraction and LC-MS/MS

50µg of protein homogenate was prepared from each sample and digested on top of an S-trap mini column (Protifi LLC) using sequencing grade modified trypsin (Promega V5111) at a 1:20 (trypsin: protein, µg) ratio. Samples were incubated for 2 hours at 47⁰C, follow by peptide elution using elution buffer (50mM TEAB pH 8.5,0.2% formic acid, 50% acetonitrile) and centrifugation (4,000 rpm, 1 minute, 3 cycles). Eluted peptides were stored at -20 ⁰C until being aliquoted for LC-MS/MS analysis.

Samples were analysed by LC−MS/MS using an Ultimate 3000 Rapid Separation LC (RSLC) nano LC system (Thermo Corporation) coupled with an Exploris 480 Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, U.S.A.). 1ug of peptide mixture was loaded, first onto an Acclaim PepMap100 C18 LC Column (5 mm Å~ 0.3 mm i.d., 5 μm, 100 Å, Thermo Fisher Scientific) at a flow rate of 10 μL min−1 maintained at 45 °C and then then separated on a 75μmx50cm C18 column (Thermo EasySpray) with an integrated emitter, using a 120min gradient from 97 % A (0.1% FA in 3% DMSO) and 3% B (0.1% FA in 80% ACN 3% DMSO), to 35 % B, at a flow rate of 250 nL min−1. The separated peptides were then injected into the mass spectrometer via EasySpray source at the Ion Transfer Tube temperature of 280oC, spray voltage 1900V and analysed using data independent (DIA) acquisition. The total LCMS run time was 150min. For the full scan mode, the MS resolution was set to 60000, with a normalized automatic gain control (AGC) target of 100%, Dynamic maximum injection time and scan range of 410−1010 m/z. DIA MSMS were acquired with 45, variable size windows covering 390-1183 m/z range, at 15000 resolution, AGC target set to 1000%, dynamic maximum injection time and normalized collision energy level of 30%.

LC-MS/MS Data Processing

Raw MS data acquired with DIA mode were processed using DIA-NN (version 1.8). Uniprot Human proteome (UP000005640, version 28.10.2021) was used as protein sequence database searches. Maximum of two missed cleavage and a maximum of 5 variable modifications per peptide were allowed. Variable modifications included acetylation at N-termini and methionine oxidation. Cysteine carboamidomethylation was used as a fixed modification. Peptide length range was set at 7-30 amino acids, precursor m/z 300-1800, fragment m/z 200-1800, precursor charge 1-4. Quantification strategy was set to AnyLC (high accuracy), FDR<1%. Finally, the data analyses were done in library-free mode (Demichev et al., 2019).