Proteoform Detection Across 17,000 Single Nuclei From Differentiating Skin Keratinocytes.

Advances in single-nucleus RNA sequencing have demonstrated advantages over single-cell transcriptomics by enabling capture of dynamic cellular states and transcriptional activity in rare or difficult-to-isolate cell types. To achieve analogous enhancements in intact protein profiling, we applied single-cell proteoform imaging mass spectrometry (scPiMS) to nuclei for label-free, proteoform-resolved analysis of >104 human skin cells. Using scPiMS, we surveyed ∼400 proteoforms across over 17,000 single nuclei isolated from human epidermal keratinocytes spanning undifferentiated (UD) proliferative to differentiated (DF) states, generating single-nucleus proteoform maps of epidermal differentiation. Proteoforms were assigned using intact mass tag matching to a reference proteoform library generated from bulk top-down LC-MS/MS analyses of UD and DF nuclei and subsequently applied to single-nucleus proteoform assignment scores. Unsupervised clustering of single-nucleus proteoform imaging mass spectrometry (snPiMS) data resolved 12 distinct proteoform-defined clusters spanning a continuum from UD proliferative states to terminal differentiation. A progenitor-enriched cluster (cluster 4) exhibited elevated high-mobility group-17, acetylated H2A/H2B variants, and H2A.Z, consistent with open and developmentally poised chromatin. An early differentiating population (cluster 3) was marked by histone H3 bearing H3K4 acetylation together with H3K9 monomethylation, indicative of transcriptional activation. Progressive histone H4 methylation (H4K20me1, H4K20me2, and H4K20me3) reflected cell cycle-coupled modification of newly synthesized H4 in progenitor-like nuclei prior to differentiation, corresponding to clusters 5, 2, and 8, respectively. Cluster 1 was enriched in H3 proteoforms bearing H3K4me3 together with H3K9me1, consistent with an active yet transcriptionally poised chromatin state. Differentiation-committed states (cluster 0) exhibited H3 proteoforms containing H3K4 and H3K9 acetylation together with H3K36me2 reflecting transcriptionally active chromatin. Bulk histone post-translational modification profiling corroborated these trends, with UD nuclei enriched in H3K9me2 and H4K20me1 and DF nuclei enriched in H4K20me2, H3K79me2, H3K27me2/3, and H3K36me3. Together, snPiMS uniquely resolves combinatorial histone proteoforms within individual nuclei, revealing a continuous chromatin trajectory across epidermal differentiation.