Tmt eleven plex reagents

Urine samples were analysed at the University of Bristol Proteomics Facility. 190 μl of urine was digested with trypsin (1.25 μg trypsin; 37 °C, overnight), labelled with Tandem Mass Tag (TMT) eleven plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, Loughborough, UK) and the labelled samples pooled. The pooled sample was desalted using a SepPak cartridge according to the manufacturer’s instructions (Waters, Milford, Massachusetts, USA). Eluate from the SepPak cartridge was evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific).

In brief, 100 µL of each sample (ensuring that no sample contained more than 50 µg of protein) was digested with trypsin, labelled with Tandem Mass Tag (TMT) eleven plex reagents (Thermo Fisher Scientific, Loughborough, UK) and the labelled samples pooled. The TMT-labelled pool was fractionated using an Ultimate 3000 nano-LC system (Thermo Scientific). All spectra were acquired using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) controlled by Xcalibur 3.0 software (Thermo Scientific) and operated in data-dependent acquisition mode using an SPS-MS3 workflow. The raw data files were processed and quantified using Proteome Discoverer software v2.1 (Thermo Scientific) and searched against the UniProt Human database (downloaded October 2019: 150,786 entries). Further detail on sample processing and proteomic analysis are described in the online supplement.