A posttranslational proteomic survey of a single anatomically preserved human 20-week postconception brain

Progress in defining the proteome of the developing human brain has lagged behind our understanding of the adult human brain, primarily due to challenges in tissue acquisition and in preservation of anatomical structure during experimental processing. Single-cell transcriptomics alone is an excellent resource for defining cellular identity, but has limited capacity to trace neuronal connectivity because proteins, the active molecules in interactions, may be transported significant distances from cell bodies and their site of synthesis. There are numerous protein-mediated transient interactions between cellular elements in the developing brain, such as between migrating cortical neurons and subplate, and thalamic projections and cortical progenitors. Anatomical approaches have identified specific cell populations that interact, allowing us to characterize the transient and dynamically changing early circuits. Proteomic data generation is now essential for ligand-receptor pair prediction and validation. Upon receipt of a single, exceptionally well-preserved 20 postconception week human brain hemisphere, we conducted fine dissections of 18 anatomically distinct brain regions, including the pia mater. These samples underwent in-depth analysis of both the total and posttranslationally modified proteomes, with the aim of creating a reference resource for investigators studying this critical stage of neurodevelopment. Here, we have presented an overview of the resulting dataset, compared the proteomic profiles across regions, and highlighted examples of variable posttranslational modifications within individual proteins. As expected, non-modified protein profiles revealed substantial differences across brain regions and structures. For instance, pia mater and thalamus were enriched for proteins involved in transcription and chromatin organization, which may suggest a higher proportion of dividing cells and/or significant epigenetic regulation in these areas at this developmental stage. In contrast, the cortical and hippocampal proteome reflected active synaptogenesis and cytoskeletal remodeling. While interregional differences in phosphorylated and acetylated peptides largely mirrored those observed in the non-modified proteome with respect to gene ontology categories, the glycosylated peptidome of the pia mater was markedly distinct. This divergence is driven by the secretion of extracellular matrix proteins and the region's intimate association with the basement membrane of the pia. Finally, by integrating our proteomic data with publicly available single-cell RNA sequencing datasets from the same developmental stage, we identified high-confidence ligand-receptor pairs (e.g., L1CAM:CD9, CNTN4:PTPRG, LGALS1:ITGB1) likely involved in thalamocortical interactions.