This study investigates the proteomic consequences of a frameshift mutation in the NOS1 (neuronal nitric oxide synthase) gene in the context of Hirschsprung's disease. Label-free quantitative (LFQ) mass spectrometry was performed on formalin-fixed paraffin-embedded (FFPE) colon tissue sections from one NOS1-mutant patient (3 technical replicates) and three control individuals. The study comprises two experimental arms: (1) total proteome profiling of all six samples, and (2) SDS-PAGE gel-band fractionation targeting proteins at ~55 kDa and ~160 kDa (migrating and narrow segments) for the three control samples. Raw data were acquired on a Thermo Fisher Orbitrap mass spectrometer and searched against the UniProt human reference proteome (UP000005640) using MaxQuant. A total of 2,193 proteins were quantified after QC filtering. Differential analysis (proDA, limma) identified 67 significantly altered proteins (adj.P<0.05, |log₂FC|>0.5; 25 upregulated, 42 downregulated) in the NOS1 mutant versus controls. NOS1 protein itself was not detected, consistent with mutation-induced protein instability or degradation. This dataset provides the first proteomic characterization of NOS1 loss-of-function in human colon tissue and complements parallel transcriptomic (RNA-seq, Iso-Seq) and immune profiling (flow cytometry) datasets from the same NOS1 mutation cohort.