*3.5. LC-MRM*/*MS Validation of Candidate Plasma Proteins Predictive of Early Response*

The differentially abundant ten proteins that were commonly significant between two groups at baseline, early treatment phase (from baseline to 1 week; Mann–Whitney U test: *p*-value < 0.05) and on the response/time interaction in LMM were selected (Figure 4A) and validated by serial isotope dilute-MRM/MS [47]. Among them, surrogate peptides were chosen by criteria except for CFHR2, which had no reliable peptide [48,49].

**Figure 4.** Liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) validation of ten candidate predictive biomarkers of early-drug response. (**A**) Venn diagram for detection of candidate biomarkers by three statistical analyses (T0 vs. T1 and T1–T0 between two groups, and response/time terms in LMM). (**B**) Boxplot of abundance of PHOX2B, SH3BGRL3, and YWHAE at T1 vs. T0, as determined by LC-MRM/MS, in responders (blue color) and non-responders (red color). \* The asterisk identifies the adjusted *p*-values that are significant at the 0.05 level.

Based on reverse standard calibration curves (Supplementary Figure S3), 15 surrogate peptides representing nine proteins were selected for protein quantification, and a representative peptide with a strong signal for each protein was selected based on the LC-MRM/MS results (Supplementary Table S4). In the validation MRM result, comparing 19 responders and five non-responders, the MYH9 could not be quantified because the heavy-light ratio was below the limit of quantitation. Of the remaining eight proteins, the three proteins, PHOX2B, SH3BGRL3, and YWHAE, showed significant differences on baseline and week 1 in responders (Wilcoxon signed-rank test: FDR-adjusted *p*-value < 0.05) but not in non-responders (Wilcoxon signed-rank test: FDR-adjusted *p*-value > 0.05; Figure 4B). After 1 week, PHOX2B protein levels increased significantly, whereas SH3BGRL3 and YWHAE protein levels decreased significantly in responders; conversely, the three proteins did not show any significant changes in non-responders. By contrast, the level of the other five proteins did not differ significantly in the two groups (Supplementary Figure S4).
