Introduction
Dermatological disorders affect approximately 1 billion people globally, representing the 4th leading cause of non-fatal disease burden (1,2). The lack of personalized and precision medicine for conditions like atopic dermatitis, psoriasis, and malignant skin carcinoma exacerbates this issue(3,4). Atopic dermatitis (AD) is the most common chronic inflammatory skin disease in children (5), yet diagnosis and severity assessment still rely on clinical scoring systems with limited molecular resolution (6–8). Skin biopsies, while informative, are invasive and ethically challenging in paediatric populations. Tape stripping offers a minimally invasive alternative (9–11), but existing proteomics approaches have been limited by low protein coverage, labour-intensive protocols, and the need for multiple strips per donor (12–14).
Here we present a novel high-throughput single tape-strip proteomics workflow compatible with a 96-well plate format, enabling robust and scalable molecular profiling from minimal sample input.
Materials and Methods
The workflow was applied to a cohort of 70 paediatric donors, 49 with AD and 21 age-matched healthy controls (HC) aged 3 months to 5 years. Detected proteins were annotated using the Human Protein Atlas and mapped to cell-type marker sets. Analyses comprised principal component analysis, differential expression analysis (AD vs. HC and severity-stratified), hierarchical clustering with enrichment analysis, and integration with published tape-strip transcriptomics data.
Results
Our method quantified over 3,000 proteins spanning six orders of magnitude in abundance. Technical reproducibility was high, with coefficients of variation of 10.7% and 11.8% for mass spectrometry and sample preparation quality controls, respectively. Annotation using the Human Protein Atlas confirmed that 94% of detected proteins are expressed in skin, including well-established barrier proteins (KRT1, KRT10, FLG2) and immune mediators (IL18, S100A7, S100A8, CCL18), while the remaining 6% have not been annotated in skin according to the Human Protein Atlas. Mapping to cell-type marker sets further identified signatures of basal and suprabasal keratinocytes, Langerhans cells, macrophages, and fibroblasts, demonstrating that single tape-strip proteomics captures a rich, multi-compartment molecular snapshot of the skin.
Principal component analysis confirmed a proteome-wide gradient corresponding to disease severity along the primary axis of variation. Differential expression analysis comparing AD patients and HC identified over 1,000 significantly altered proteins. Key upregulated proteins included S100A7, S100A8, and IL18, consistent with innate immune activation, while structural keratins KRT1, KRT2, and KRT10 were significantly downregulated, reflecting impaired epidermal barrier function. Severity-stratified analysis yielded over 100 significantly altered proteins, demonstrating for the first time that clinically defined AD severity states can be proteomically resolved from single tape-strip samples.
Severity-associated proteins including IL18, SERPINB12, APOA1, APOA2, and A2M implicate inflammatory, barrier, and lipid metabolism pathways in disease progression. Integration with published tape-strip transcriptomics data showed broad concordance of disease-associated signals across modalities, while proteomics additionally identified APOA1, previously reported as dysregulated in AD circulation, as a novel tape-strip biomarker candidate not captured at the transcript level.
Hierarchical clustering and enrichment analysis of significantly altered proteins revealed coordinated biological programs, including adaptive immune and Langerhans cell signatures enriched in severe disease, proteasome-related clusters consistent with immunoproteasome involvement in AD, and keratinocyte differentiation clusters reduced with disease severity.
Discussion
This represents a notable advance, as molecular stratification of disease severity has been notoriously difficult to achieve with existing approaches. Together, these results establish single tape-strip proteomics as a powerful, non-invasive platform for molecular stratification of paediatric atopic dermatitis, with broad potential for longitudinal monitoring and biomarker discovery in inflammatory skin diseases.
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