Proteomic Analysis of Immunized Peripheral Blood

Thirty-two individuals (16 for TFP1 injection and 16 for GVHSV injection) were immunized as described above. Peripheral blood was extracted at 14 dpi, and PB-RBCs were purified by 2 density gradient centrifugations (1600 rpm, Ficoll 1.007; Lymphoprep, Reactiva, Sigma-Aldrich) as previously described [9 (link)]. The 99.9% purity of RBCs was estimated by optical microscopy (Supplementary Figure S1). Cells (10⁷ cells per individual) were pelletized by centrifugation (1600 rpm, 5 min), the supernatant was removed, and the cell pellet was washed 3 times with PBS. The pellet was then digested, cleaned-up/desalted, and pooled into 2 pools of 8 individuals for each condition (TFP1 or GVHSV) (Figure 1). Samples were subjected to liquid chromatography and mass spectrometry analysis (LC-MS) as previously described [14 (link),29 (link)].