Fig 2: Spatial proteomic gradient of human skin.a Unique and overlapping protein groups in outer and inner epidermis, dermis and subcutis skin layers (UpSet plot) One independent experiment of pooled samples (N = 1) from five donors for each layer). b Relative expression levels of important structural and immunological proteins across skin layers and in primary keratinocytes and fibroblasts. Clustering is based on the log2 expression level of the proteins in skin layers (DDA). c Protein mass percentage of keratins, collagens, glycoproteins and secreted proteins across skin layers. Percentage was calculated by dividing the summed iBAQ intensities of the proteins of interest by all summed iBAQ intensities of all proteins (see Methods). d Expression profiles of the S100A family members of proteins across skin layers, illustrating their layer-specific, functional abundance. e, f Estimated protein abundance vs protein rank highlighting keratin 1 (KRT1), keratin 14 (KRT14), and collagen III (COL3A1) (upper panels) and (g, h) immunohistochemical stainings (KRT1, brown, KRT14, red, COL3A1, brown) (lower panels), scale bar: 50 µm. KRT1 is the protein with the 7th highest estimated abundance in outer epidermis, whereas KRT14 is the 6th most abundant. COL3A1 is the 19th most abundant protein in dermis, which is also reflected in the staining. i Estimated abundance of KRT1 and (j) COL3A1 across libraries based on pooled samples of skin layers and skin-derived cells examined over one independent experiment from pooled samples (N = number of pooled donor samples). (CD1A+dendritic cells (N = 3), CD14+ dendritic cells (N = 3), MΦ; macrophages (N = 4), MC; mast cells (N = 6), Fib; fibroblast (N = 5), KC; keratinocytes (N = 5), EC; endothelial cells (N = 4), Mel; melanocytes (N = 5), skin layers (Outer Epi, Inner Epi, Dermis, Subcutis) (N = 5). Source data are provided as a Source Data file.

Fig 3: In-depth MS-based proteomic analysis of skin-associated cellular subsets.a Single-run analyses of DC; CD1A+dendritic cells (N = 3), MΦ; macrophages (N = 4), MC; mast cells (N = 6), ET; epidermal T cells (N = 6), DT; dermal T cells (N = 6), EC; endothelial cells (N = 4), Mel; melanocytes (N = 5), Fib; fibroblasts (N = 5) and KC; keratinocytes (N = 5) using data-independent (DIA) acquisition. Data are presented as mean±SEM. N represents number of biologically independent samples. The number of quantified protein groups for each major cell lineage is roughly similar. Source data are provided as a Source Data file. b Principal component analysis (PCA) of all proteomes from cellular subsets. Color code from panel (a). The PCA separates cultivated fibroblast and keratinocytes from FACS-sorted endothelial cells (EC) and melanocytes (Mel) as well as from the immune cells, as indicated by enclosing ovals. c Heatmap of protein abundances of 1272 differentially expressed proteins (ANOVA, FDR < 0.01, FCH > 2) after unsupervised hierarchical clustering. d Differentially expressed proteins in epidermal T cells vs. dermal T cells (volcano plot, FDR < 0.05, FCH > 2).

Fig 4: In-depth MS-based proteomic analysis of skin layers and its cellular subsets.a Workflow for measuring the skin proteome including skin layers, cultivated keratinocytes, and fibroblasts and FACS-sorted melanocytes, endothelial cells, and immune cells. b High-resolution MS analyses of fractionated, pooled samples in data-dependent acquisition mode (DDA) and subsequent (c) single-run analyses of three to six biological replicates in data-independent (DIA) acquisition mode. d Number of protein groups for each major cell subset and skin layers based on pooled samples (one independent experiment) for cell subsets and skin layers, respectively examined over one independent experiment (CD1A+dendritic cells (N = 3), CD14+ dendritic cells (N = 3), macrophages (N = 4), mast cells (N = 6), fibroblast (N = 5), keratinocytes (N = 5), endothelial cells (N = 4), melanocytes (N = 5), skin layers (N = 5). A total of 10,701 protein were identified. e Principal component analysis (PCA) of all proteomes from skin layers, primary cells and immune cell types. Color code from panel (d). The fractionated proteome of PBMC (green dot) was included as library for skin-associated T cells. Component 1 and 2 account for 24 and 11% of the total data variation, respectively.

Fig 5: The proteomes of cultivated fibroblasts and keratinocytes.a MS-intensity (iBAQ) of endosialin across cell types (left upper panel) compared to its immunohistochemical staining obtained from the Human Protein Atlas (Scale bar: 100 µm, see Methods and Supplementary Data 17). b Protein abundance profiles of the top 100 proteins across cell types with the most similar fibroblast-enriched profile as endosialin (reference profile, blue line). Each line represents one protein. c MS-intensity of selected fibroblast-enriched proteins (GREM1, Col11A1, c12orf75,) across cell types. d Protein expression profiles of the top 100 proteins across cell types with the most similar keratinocyte-enriched profile as KLK10 (blue line). e iBAQ values of the keratinocyte-enriched protein TMEM40 across cell types and its immunohistochemical staining obtained from the Human Protein Atlas (Scale bar: 100 µm, see Methods and Supplementary Table 17). f MS-intensity of selected keratinocyte-enriched proteins (ANXA8L1, EGFLAM, FGFBP1) across cell types. All barplots show estimated intensity (data are presented as mean±SEM (IBAQ, log2)). DC; CD1A+dendritic cells (N = 3), MΦ; macrophages (N = 4), MC; mast cells (N = 6), DT; dermal T cells (N = 6), ET; epidermal T cells (N = 6), EC; endothelial cells (N = 4), Mel; melanocytes (N = 5), Fib; fibroblasts (N = 5), KC; keratinocytes (N = 5). Source data are provided as a Source Data file.