Multiplexed Proteomics Workflow with TMTpro

For proteomics analysis, samples were boiled in SDS buffer and subjected to SP3-based cleanup and tryptic digest (64 (link)). To enable multiplexing, samples were labeled with TMTpro 16-plex reagents (Thermo Fisher Scientific) (65 (link)). A total of 5 μg peptide/sample was assigned to channels 1–12. Pooled plexes were subjected to high-pH HPLC separation into 24 fractions. An estimate of 1 μg each fraction was measured on a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). For database search, MaxQuant version 1.6.10.43 (66 (link)) was used while enabling TMTpro 16-plex reporter ion quantitation with a PIF setting of 0.5. Downstream analysis was done with R. For quantitation, a minimum of 75% valid TMT reporter ion intensities was required. The remaining missing values were imputed by employing k-nearest neighbor algorithm. Corrected reporter ion intensities were normalized against the internal reference sample and scaled using median-median absolute deviation (median-MAD) normalization. For significance calling 2-sample moderated, 2-tailed Student’s t testing as well as moderated F testing was applied (limma R package) (67 (link)). P values were adjusted using the Benjamini-Hochberg method.