Tmtpro 16plex reagents

Proteins were extracted using an EasyPep sample preparation kit (Pierce, San Jose, CA, USA) using manufacturer recommendations. The proteins were quantified using the Micro BCA kit method (Pierce, San Jose, CA, USA).
Twenty-five micrograms of proteins of each sample were digested and labeled with TMTpro™ 16-plex reagents (Thermo Fisher Scientific, San Jose, CA, USA), mixed in equimolar amounts. A fractionation was purchased using a High pH Reversed-Phase Peptide Fractionation Kit (Pierce, San Jose, CA, USA), according to manufacturer recommendations. The tryptic peptide solutions were dried under vacuum and reconstituted in 20 µL water/1% formic acid (v/v) each.

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.

Samples were reconstituted in TEAB to 1 mg/ml, then labeled with TMTpro 16plex reagents (Thermo Fisher) following the manufacturers protocol. Briefly, TMTpro tags were removed from the −20°C freezer and allowed to come to room temperature, after which acetonitrile was added. Individual TMT tags were added to respective samples, and the reaction was allowed to occur at room temperature for 1 h. 5% hydroxylamine was added to quench the reaction, after which the samples for each experiment were combined into a single tube. Since we performed quantitation on the unlabeled peptides, 0 day samples were added to four of the unused channels, increasing the signal for the unlabeled peptides. TMTpro‐tagged samples were frozen, dried down in the Speed Vac, and then desalted using homemade C18 spin columns to remove excess tag prior to high pH fractionation.