(A) Workflow for dMS sample processing and instrumental analysis. Step 1: deparaffinization, antigen retrieval, and lysis. A total of 10 μL from each sample (n = 23) was combined and aliquoted into 9 technical replicates. Step 2: FASP digestion. Each sample was normalized to 30 μg. A total of 750 fmol of ovalbumin was added as an internal standard. A pooled instrument control was made by combining 5 μL from each sample (n = 32). Samples (n = 33) were reordered. Step 3: nLC-MS/MS analysis. Injection of ~0.2 μg on to C18 Picochip column Orbitrap Velos Pro and analysis. (B) Schematic illustration of MCV T antigen transcripts. Small T (yellow, Frame 1) and Large T (yellow, Frame 1; orange, Frame 3) transcripts from the early region including start, splice, and termination sites are shown. Both small T and large T encode DnaJ domain. Small T and MCV unique domains, origin binding (OBD), zinc finger, ATPase, and helicase domains are depicted. The location of mutations and deletions found in MCC tumor large T are highlighted with a gray line. Positions of the 4 MCV peptides identified by dMS analysis are indicated with green, orange, purple, and blue arrows. (C) Dot plots for the relative abundance of identified viral peptides in MCV⁺ (red, n = 12) versus negative (blue, n = 11) MCC samples. Peptides and their rankings (Table 1) are shown in green (ID#14), orange (ID#4), purple (ID#15), and blue (ID#1). Data are shown as mean ± SD. P values were based on 2-sided equal variance Student’s t test.