Figure 2. Protein identification and validation using the SASI approach.

(A) Distribution of relative abundance of proteins precipitated with antibodies. Three biological experiments (denoted as Patients #1, #3 and #6) were performed, each quantitatively identifying about 800 to 900 proteins differentially precipitated between pre-vaccination and post-vaccination. Only a small fraction of proteins were observed to induce a highly differential antibody response after vaccination, indicating that most of antibodies may still target similar epitopes regardless of vaccine. (B) Pearson correlation. Pair-wise comparison of proteins observed in common showed higher similar pattern as evident by Pearson correlation (0.75, 0.85 and 0.86 for Patients #1 vs #6, #3 vs #6 and #1 vs #6, respectively). (C) A representative MS/MS spectrum. A peptide (VAVNDAHLLQYNHR) derived by trypsin protease from galectin-3 was identified and spectral assignment was shown. (D) Confirmation of the quantitative mass spectrometry (MS) results. Five molecules detected by the quantitative MS were used for the Western blot analysis. Pre- and post-vaccination antibodies of patient 6 (also used for SASI screen) with favorable outcome (DFS > 3 years) were used for immunoprecipitation. The precipitate was separated by SDS-PAGE followed by western blot using antibodies against the indicated proteins. The fold-change as detected by mass spectrometry in the SASI screen are shown to the right of each blot and correlates well with the qualitative results in the western blot, confirming that the MS data are all in keeping with the orthogonal method.