Tmt 10 plex isobaric reagents

Tandem mass tag (TMT) 10-plex isobaric reagents, bicinchoninic acid (BCA) assay kit - reducing agent compatible, tris (2-carboxyethyl) phosphine (TCEP), and LC/MS-grade solvents such as acetone, acetonitrile (ACN), and water were purchased from Thermo Fisher Scientific (Waltham, MA). Other reagents and materials were purchased from the following companies: Dithiothreitol (DTT) and urea from AMRESCO (Solon, OH), Sodium dodecyl sulfate (SDS), Trizma base from USB (Cleveland, OH) and sequencing-grade modified trypsin from Promega Corporation (Madison, WI), POROS20 R2 bead from Applied Biosystems (Foster City, CA). High-purity (>97%) mass spectrometry (MS) grade ovalbumin from Protea (Morgantown, WV), HLB OASIS column from Waters (Milford, MA). All other reagents, unless noted, were purchased from Sigma-Aldrich (St. Louis, MO).

The proteomic analysis was performed using group-wise pooled samples (analysis 1). In order to validate pooling, proteomic analysis was also performed for individual (non-pooled) plasma samples (analysis 2) from irradiated and control groups of female and male juvenile black mice, i.e. for four groups. A comprehensive description of sample preparation procedures is supplied in SI Appendix, Supplemental Information Text S1. In summary, analysis 1 was prepared by mixing equal volumes of plasma from each sample in a group to achieve representative pooled samples. The global pooled reference sample was prepared by mixing equal volumes from all samples. Analysis 2 was prepared by mixing equal volumes from the sample that belonged to the respective groups. Immunodepletion was performed using the Seppro mouse spin column kit (SEP110; Sigma-Aldrich; St Louis, MO, USA) according to the manufacturer’s instruction. Subsequently, the samples were processed according to the modified filter-aided sample preparation (FASP) method^{59 (link)}. The immunodepleted plasma samples were processed and the resulting peptides were labelled using the TMT 10plex isobaric reagents according to manufacturer’s instructions (Thermo Scientific).