Molecular Pixelation (MPX) is a DNA-based method for spatial proteomics of single cells, using antibody-oligonucleotide conjugates (AOCs) and DNA-based molecular pixels. The method enables the spatial mapping of proteins on single cells by sequentially associating AOCs into local neighborhoods using unique molecular identifiers (UMIs), forming over 1,000 spatially connected zones per cell in 3D. For each single cell, DNA sequencing reads are arranged into spatial proteomics networks for 76 proteins. By analyzing immune cell dynamics using spatial statistics on graph representations of the data, MPX identifies known and new patterns of protein spatial organization on chemokine-stimulated T cells, highlighting its potential in defining cell states by protein spatial arrangement. MPX uses DNA-tagged AOCs bound to their protein targets on chemically fixed cells to survey cell surface protein arrangement in a highly multiplexed manner. The assay is performed without sample immobilization or single-cell compartmentalization, in a standard reaction tube. Spatial analysis of protein arrangement is enabled by forming two associations between spatially proximate AOCs into local neighborhoods through the incorporation of a unique molecular identifier (UMI), similar to proximity barcoding. The generated amplicons are sequenced and spatial relationships of proteins are inferred from graph representations of the data for each single cell. AOCs bound to cells are associated into local neighborhoods using DNA pixels, which are single-stranded DNA molecules with a diameter of <100 nm. Each DNA pixel contains a unique pixel identifier (UPI) and is generated by rolling circle amplification from circular DNA templates. Once added to the reaction, each DNA pixel can hybridize to multiple AOC molecules in proximity on the cell surface. The UPI sequence of the hybridized DNA pixel is then incorporated onto the AOC oligonucleotide by a gap-fill ligation reaction, creating neighborhoods where the set of AOCs within each neighborhood share the same UPI sequence. Following enzymatic degradation of the first DNA pixel set, a second set of DNA pixels is similarly incorporated by hybridization and gap-fill ligation reactions. The generated amplicons are then amplified by PCR and sequenced. Each sequenced molecule contains four distinct DNA barcode motifs; a UMI to enable identification of unique AOC molecules, a protein identity barcode and two UPI barcodes with neighborhood memberships. The relative location of each unique AOC molecule can be inferred from the overlap of UPI neighborhoods created from the two serial DNA pixel hybridization and gap-fill ligation steps. Each sequenced unique molecule can be represented as an edge in a bipartite graph, with UPI-A and UPI-B sequences as nodes and protein identity as edge attributes, or as a one-mode projected graph of UPI-A sequences as nodes and protein identities as node attributes. The graphs generated from a sequenced sample following data processing and filtering contain graph components thatMolecular Pixelation (MPX) is a DNA-based method for spatial proteomics of single cells, using antibody-oligonucleotide conjugates (AOCs) and DNA-based molecular pixels. The method enables the spatial mapping of proteins on single cells by sequentially associating AOCs into local neighborhoods using unique molecular identifiers (UMIs), forming over 1,000 spatially connected zones per cell in 3D. For each single cell, DNA sequencing reads are arranged into spatial proteomics networks for 76 proteins. By analyzing immune cell dynamics using spatial statistics on graph representations of the data, MPX identifies known and new patterns of protein spatial organization on chemokine-stimulated T cells, highlighting its potential in defining cell states by protein spatial arrangement. MPX uses DNA-tagged AOCs bound to their protein targets on chemically fixed cells to survey cell surface protein arrangement in a highly multiplexed manner. The assay is performed without sample immobilization or single-cell compartmentalization, in a standard reaction tube. Spatial analysis of protein arrangement is enabled by forming two associations between spatially proximate AOCs into local neighborhoods through the incorporation of a unique molecular identifier (UMI), similar to proximity barcoding. The generated amplicons are sequenced and spatial relationships of proteins are inferred from graph representations of the data for each single cell. AOCs bound to cells are associated into local neighborhoods using DNA pixels, which are single-stranded DNA molecules with a diameter of <100 nm. Each DNA pixel contains a unique pixel identifier (UPI) and is generated by rolling circle amplification from circular DNA templates. Once added to the reaction, each DNA pixel can hybridize to multiple AOC molecules in proximity on the cell surface. The UPI sequence of the hybridized DNA pixel is then incorporated onto the AOC oligonucleotide by a gap-fill ligation reaction, creating neighborhoods where the set of AOCs within each neighborhood share the same UPI sequence. Following enzymatic degradation of the first DNA pixel set, a second set of DNA pixels is similarly incorporated by hybridization and gap-fill ligation reactions. The generated amplicons are then amplified by PCR and sequenced. Each sequenced molecule contains four distinct DNA barcode motifs; a UMI to enable identification of unique AOC molecules, a protein identity barcode and two UPI barcodes with neighborhood memberships. The relative location of each unique AOC molecule can be inferred from the overlap of UPI neighborhoods created from the two serial DNA pixel hybridization and gap-fill ligation steps. Each sequenced unique molecule can be represented as an edge in a bipartite graph, with UPI-A and UPI-B sequences as nodes and protein identity as edge attributes, or as a one-mode projected graph of UPI-A sequences as nodes and protein identities as node attributes. The graphs generated from a sequenced sample following data processing and filtering contain graph components that