Higher-Throughput Processing of Proteoforms from Single Cells and Nuclei from Tumors via Arrayed Deposition

Single-cell proteomics (SCP) has transformed the study of cellular heterogeneity, yet intact proteoform-level analysis at single-cell resolution has remained technically constrained by throughput limitations and reliance on experiment-specific reference databases. Here, we present a series of integrated advances to single-cell proteoform imaging mass spectrometry (scPiMS) that substantially expand its throughput, analytical depth, and applicability to primary human tissue. Replacing stochastic dropcasting with image-guided cell arraying using the cellenONE platform increases productive cellograms from 1,400 to 5,500 per day. This reflects a four-fold improvement without compromising single-cell proteoform extraction quality. Through co-arraying of single cells and isolated nuclei from the same sample on one slide, we enable simultaneous profiling of cytoplasmic and nuclear proteoform compartments in one acquisition, with relative quantification. This recovers complementary proteoform landscapes that together increase the total coverage relative to either cellular fraction alone. A multistage database identification strategy combining abundance-scaled bottom-up databases, sample-specific top-down data, and a data-dependent score to filter SwissProt searches. This combinatorial approach prefilters ambiguous proteoforms to high-confidence hits for manual validation with ∼ 50% confirmation rates. Applied to freshly resected meningioma tissue from four patients spanning WHO grades 1 and 2, scPiMS identifies biologically coherent pathway signatures including amyloid and structural remodeling, receptor kinase oncogenic signaling, and epigenetic dysregulation. Together, these advances position scPiMS as a scalable platform to resolve proteoform-level biology in complex primary tissues with initial application to meningioma, revealing patient-specific enrichment of RAS/MAPK signaling components, epigenomic dysregulation, and receptor tyrosine kinase (RTK) signaling heterogeneity at single-cell and single-nucleus resolution.