Proteoform Detection across 17000 Single Nuclei from Differentiating Skin Keratinocytes.

Area of Science:

• Proteomics
• Cell Biology
• Epigenetics

Background:

• Single-nucleus RNA sequencing (snRNA-seq) offers advantages over single-cell transcriptomics for capturing dynamic cellular states.
• Analogous protein profiling enhancements are needed for a comprehensive understanding of cellular dynamics.

Purpose of the Study:

• To apply single-cell Proteoform imaging Mass Spectrometry (scPiMS) for label-free, proteoform-resolved analysis of human skin cell nuclei.
• To generate single-nucleus proteoform maps of epidermal differentiation and resolve chromatin trajectories.

Main Methods:

• scPiMS was applied to >10^4 human epidermal keratinocyte nuclei.
• Proteoforms were assigned using intact mass tag (IMT) matching to a reference library and Proteoform Assignment Scores (PAScores).
• Unsupervised clustering of single-nucleus PiMS (snPiMS) data resolved 12 distinct proteoform-defined clusters.

Main Results:

• scPiMS surveyed ~400 proteoforms across >17,000 single nuclei, mapping epidermal differentiation.
• 12 distinct clusters revealed a continuum from undifferentiated to terminally differentiated states.
• Specific histone proteoform modifications (e.g., H3K4 acetylation, H4K20 methylation) correlated with distinct differentiation stages and chromatin states.

Conclusions:

• snPiMS uniquely resolves combinatorial histone proteoforms within individual nuclei.
• The study reveals a continuous chromatin trajectory across epidermal differentiation.
• scPiMS provides unprecedented resolution for studying epigenetic regulation in cellular differentiation.