Protein misfolding diseases, including α1-antitrypsin deficiency (AATD), pose substantial health challenges, with their cellular progression still poorly understood1-3. We use spatial proteomics by mass spectrometry and machine learning to map AATD in human liver tissue. Combining Deep Visual Proteomics (DVP) with single-cell analysis4,5, we probe intact patient biopsies to resolve molecular events during hepatocyte stress in pseudotime across fibrosis stages. We achieve proteome depth of up to 4,300 proteins from one-third of a single cell in formalin-fixed, paraffin-embedded tissue. This dataset reveals a potentially clinically actionable peroxisomal upregulation that precedes the canonical unfolded protein response. Our single-cell proteomics data show α1-antitrypsin accumulation is largely cell-intrinsic, with minimal stress propagation between hepatocytes. We integrated proteomic data with artificial intelligence-guided image-based phenotyping across several disease stages, revealing a late-stage hepatocyte phenotype characterized by globular protein aggregates and distinct proteomic signatures, notably including elevated TNFSF10 (also known as TRAIL) amounts. This phenotype may represent a critical disease progression stage. Our study offers new insights into AATD pathogenesis and introduces a powerful methodology for high-resolution, in situ proteomic analysis of complex tissues. This approach holds potential to unravel molecular mechanisms in various protein misfolding disorders, setting a new standard for understanding disease progression at the single-cell level in human tissue.

Clinical and Transplant Pathology Centre Institute for Clinical and Experimental Medicine Prague Czech Republic

Danish Institute of Advanced Study University of Southern Denmark Odense Denmark

Department of Clinical Research Faculty of Health Sciences University of Southern Denmark Odense Denmark

Department of Gastroenterology and Hepatology Centre for Liver Research Odense Denmark

Department of Hepatogastroenterology Institute for Clinical and Experimental Medicine Prague Czech Republic

Department of Pathology and Molecular Medicine 3rd Faculty of Medicine Charles University and Thomayer Hospital Prague Czech Republic

Department of Pathology Odense University Hospital Odense Denmark

Department of Proteomics and Signal Transduction Max Planck Institute of Biochemistry Martinsried Germany

Gene Center and Department of Biochemistry Ludwig Maximilians Universität München Munich Germany

Institute of Pathology University Hospital Aachen RWTH Aachen University Aachen Germany

Medical Clinic 3 Gastroenterology Metabolic Diseases and Intensive Care University Hospital RWTH AachenHealth Care Provider of the European Reference Network on Rare Liver Disorders Aachen Germany

NNF Center for Protein Research Faculty of Health Sciences University of Copenhagen Copenhagen Denmark

Zobrazit více v PubMed

Greene, C. M. et al. α1-Antitrypsin deficiency. PubMed

Strnad, P., McElvaney, N. G. & Lomas, D. A. Alpha1-antitrypsin deficiency. PubMed

Hipp, M. S., Park, S.-H. & Hartl, F. U. Proteostasis impairment in protein-misfolding and -aggregation diseases. PubMed

Mund, A. et al. Deep Visual Proteomics defines single-cell identity and heterogeneity. PubMed PMC

Rosenberger, F. A. et al. Spatial single-cell mass spectrometry defines zonation of the hepatocyte proteome. PubMed PMC

Petrosius, V. et al. Evaluating the capabilities of the Astral mass analyzer for single-cell proteomics. Preprint at

Guzman, U. H. et al. Ultra-fast label-free quantification and comprehensive proteome coverage with narrow-window data-independent acquisition. PubMed PMC

Guo, T., Steen, J. A. & Mann, M. Mass-spectrometry-based proteomics: from single cells to clinical applications. PubMed

Nordmann, T. M. et al. Spatial proteomics identifies JAKi as treatment for a lethal skin disease. PubMed PMC

Chiti, F. & Dobson, C. M. Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade. PubMed

Selkoe, D. J. & Hardy, J. The amyloid hypothesis of Alzheimer’s disease at 25 years. PubMed PMC

Goedert, M., Jakes, R. & Spillantini, M. G. The synucleinopathies: twenty years on. PubMed PMC

Lomas, D. A., Evans, D. L., Finch, J. T. & Carrell, R. W. The mechanism of Z alpha 1-antitrypsin accumulation in the liver. PubMed

Brantly, M., Nukiwa, T. & Crystal, R. G. Molecular basis of alpha-1-antitrypsin deficiency. PubMed

Clark, V. C. et al. Clinical and histologic features of adults with alpha-1 antitrypsin deficiency in a non-cirrhotic cohort. PubMed

Lindblad, D., Blomenkamp, K. & Teckman, J. Alpha-1-antitrypsin mutant Z protein content in individual hepatocytes correlates with cell death in a mouse model. PubMed

Rudnick, D. A. et al. Analyses of hepatocellular proliferation in a mouse model of alpha-1-antitrypsin deficiency. PubMed

Chambers, J. E. et al. Z-α1-antitrypsin polymers impose molecular filtration in the endoplasmic reticulum after undergoing phase transition to a solid state. PubMed PMC

Segeritz, C.-P. et al. hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in α1-antitrypsin deficiency. PubMed PMC

Fromme, M., Schneider, C. V., Trautwein, C., Brunetti-Pierri, N. & Strnad, P. Alpha-1 antitrypsin deficiency: a re-surfacing adult liver disorder. PubMed

Zhang, Y. et al. LMAN1-MCFD2 complex is a cargo receptor for the ER-Golgi transport of α1-antitrypsin. PubMed PMC

Schmidt, B. Z. & Perlmutter, D. H. Grp78, Grp94, and Grp170 interact with alpha1-antitrypsin mutants that are retained in the endoplasmic reticulum. PubMed

Werder, R. B. et al. Adenine base editing reduces misfolded protein accumulation and toxicity in alpha-1 antitrypsin deficient patient iPSC-hepatocytes. PubMed PMC

Spivak, I. et al. Alpha‐1 antitrypsin inclusions sequester GRP78 in a bile acid–inducible manner. PubMed PMC

Niu, L. et al. Noninvasive proteomic biomarkers for alcohol-related liver disease. PubMed PMC

Cho, S.-H. et al. Lgals3bp suppresses colon inflammation and tumorigenesis through the downregulation of TAK1-NF-κB signaling. PubMed PMC

Khodayari, N. et al. Characterization of hepatic inflammatory changes in a C57BL/6J mouse model of alpha1-antitrypsin deficiency. PubMed

Porat-Shliom, N. Compartmentalization, cooperation, and communication: the 3Cs of hepatocyte zonation. PubMed PMC

Piccolo, P. et al. Down‐regulation of hepatocyte nuclear factor‐4α and defective zonation in livers expressing mutant Z α1‐antitrypsin. PubMed

Crowther, D. C. et al. Practical genetics: alpha-1-antitrypsin deficiency and the serpinopathies. PubMed

Liu, Z. et al. A ConvNet for the 2020s. Preprint at https://arxiv.org/abs/2201.03545v2 (2022).

Yang, C. et al. Increased expression of epidermal growth factor-like domain-containing protein 7 is predictive of poor prognosis in patients with hepatocellular carcinoma. PubMed

Carlson, J. A. et al. Accumulation of PiZ alpha 1-antitrypsin causes liver damage in transgenic mice. PubMed PMC

Yusa, K. et al. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells. PubMed PMC

Hidvegi, T., Schmidt, B. Z., Hale, P. & Perlmutter, D. H. Accumulation of mutant alpha1-antitrypsin Z in the endoplasmic reticulum activates caspases-4 and -12, NFkappaB, and BAP31 but not the unfolded protein response. PubMed

Rosenberger, F. A., Thielert, M. & Mann, M. Making single-cell proteomics biologically relevant. PubMed

Coscia, F. et al. A streamlined mass spectrometry–based proteomics workflow for large-scale FFPE tissue analysis. PubMed

Fan, R. Integrative spatial protein profiling with multi-omics. PubMed

Henrich, M. T. et al. Determinants of seeding and spreading of α-synuclein pathology in the brain. PubMed PMC

Bassil, F. et al. Amyloid-beta (Aβ) plaques promote seeding and spreading of alpha-synuclein and tau in a mouse model of Lewy body disorders with Aβ pathology. PubMed PMC

Schneider, C. V. et al. Liver Phenotypes of European Adults Heterozygous or Homozygous for Pi∗Z Variant of AAT (Pi∗MZ vs Pi∗ZZ genotype) and Noncarriers. PubMed

Stringer, C., Wang, T., Michaelos, M. & Pachitariu, M. Cellpose: a generalist algorithm for cellular segmentation. PubMed

Wolf, T. et al. HuggingFace’s transformers: state-of-the-art natural language processing. Preprint at https://arxiv.org/abs/1910.03771v5 (2020).

McInnes, L., Healy, J. & Melville, J. UMAP: uniform manifold approximation and projection for dimension reduction. Preprint at https://arxiv.org/abs/1802.03426v3 (2020).

Pedregosa, F. et al. Scikit-learn: machine learning in Python.

Schmacke, N. A. et al. SPARCS, a platform for genome-scale CRISPR screening for spatial cellular phenotypes. Preprint at

Thielert, M., Weiss, C. A. M., Mann, M. & Rosenberger, F. A. in

Demichev, V., Messner, C. B., Vernardis, S. I., Lilley, K. S. & Ralser, M. DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. PubMed PMC

Ammar, C., Schessner, J. P., Willems, S., Michaelis, A. C. & Mann, M. Accurate label-free quantification by directLFQ to compare unlimited numbers of proteomes. PubMed PMC

Elizarraras, J. M. et al. WebGestalt 2024: faster gene set analysis and new support for metabolomics and multi-omics. PubMed PMC

Szklarczyk, D. et al. The STRING database in 2023: protein–protein association networks and functional enrichment analyses for any sequenced genome of interest. PubMed PMC

Uhlén, M. et al. The human secretome. PubMed

Perez-Riverol, Y. et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. PubMed PMC

Sarkans, U. et al. The BioStudies database—one stop shop for all data supporting a life sciences study. PubMed PMC