Proteome Analysis Using TMTpro 16plex

The proteome analysis was performed similar as previously described (Shen et al., 2020 (link)). Digested peptides were cleaned-up and labeled using TMTpro 16plex label reagents (Thermo Fisher Scientific, San Jose, USA). Peptides were separated into 30 fractions, which were later combined into 15 fractions. Subsequently, the fractions were dried, redissolved in 2% ACN/0.1% formic acid. All the samples were analyzed using liquid chromatography (LC)-coupled tandem mass spectrometry (MS/MS) with a data-dependent acquisition mode on an Orbitrap 480 (Thermo Fisher Scientific, San Jose, USA). During each acquisition, peptides were analyzed using a 30 min-long LC gradient (from 7 to 30% buffer B). The m/z range of MS1 was 375–1,800, with a resolution of 60,000, normalized Automatic Gain Control (AGC) target of 300%, maximum ion injection time (max IT) of 50 ms, and compensation voltages of −48 V and −68 V for FAIMS Pro™. MS/MS experiments were performed with a resolution of 30,000, normalized AGC target of 200%, and 86 ms max IT for Serum and 100 ms for PBMC. The turbo-TMT and the advanced peak determination were enabled.