RAW LC-MSMS proteomics data of affinity proteomics conducted using DNA baits treated with human neuroblastoma cells nuclear extract

N 6 -methyldeoxyadenosine (N 6 medA) is an endogenous DNA modification found across diverseorganisms, but its abundance, genomic distribution, and biological significance in mammalsremain poorly defined. It’s extremely low abundance in mammalian DNA (< 1 per 10 millionadenines) presents a major analytical challenge. Here, we developed and validated anultrasensitive isotope-dilution nano liquid chromatography–nanospray ionization Orbitrap massspectrometry method for accurate quantification of N 6 medA in genomic DNA. Analysis ofhuman prefrontal cortex samples from individuals aged 18–91 years revealed a significantlinear increase in genomic N 6 medA abundance with chronological age. N 6 medA levels alsoshowed a trend toward elevation in individuals with mild cognitive impairment (MCI) andAlzheimer’s disease (AD) compared with age-matched controls. Genome-wide profiling usingNAME-Seq and MeDIP-Seq identified age-associated changes in N 6 medA and genomicregions differentially methylated in MCI and AD. Cross-validated N 6 medA sites were enrichednear genes involved in neuronal function, including glutamatergic synaptic signaling, axonguidance, and long-term depression. Photoaffinity proteomics identified candidate N 6 medAreader proteins, including YTHDF2, WDR3, ZHX1, and HNRNPC with roles in DNA repair,axonal transport, and synaptic signaling. Together, our findings establish N 6 medA as an age-associated DNA modification in the human brain and support its potential involvement in brainaging and neurodegeneration.