Single-cell Deep Visual Proteomics (scDVP) enables the resolution of the context-dependent, spatial proteome of single cells in intact tissue across biological conditions and diseases. In this study, we focus on alpha1-antitrypsin deficiency (AATD), a genetic disorder caused by an autosomal, co-dominant inherited mutation in the SERPINA1 gene. Misfolded alpha1-antitrypsin (AAT) protein accumulates within hepatocytes, which induces cellular stress and ultimately leads to liver fibrosis and cirrhosis in affected individuals. As these liver complications often advance silently and disease manifestation differs greatly across affected individuals, timely detection and management of the disease are hindered. While current understanding suggests a correlation between the severity of liver damage and the extent of AAT aggregation, the precise molecular events in time and space are not well understood.

Here, we leverage the mass resolution, speed, and sensitivity of the Thermo Scientific Orbitrap Astral mass spectrometer (MS) and our Deep Visual Proteomics (DVP) workflow. Decreasing sample loss, by minimal sample transfer steps and ensuring high LC/MS sensitivity with a combination of the Evosep Whisper40 low flow gradient, Ionopticks Aurora columns and the Orbitrap Astral MS. We first acquired deep proteomes of liver hepatocytes from samples containing 10-15 hepatocytes (~100 hepatocyte shapes) each to characterize the relationship between aggregate load and hepatocyte health. We then applied single cell DVP to precisely map the proteome of individual hepatocytes in regions with striking spatial disease manifestations, such as cells with high and no load of AAT in direct proximity. This data uniquely enabled us to characterize the proteome of a single hepatocyte shape (~1/3 to 1/2 of a hepatocyte) at a depth of up to 3600 proteins. We find that the hepatocyte proteome was strongly dependent on both fibrosis stage and AAT load. We observed significant changes in the abundance of proteins produced in the ER and targeted for secretion, highlighting an unfolded protein response (UPR) involving all canonical UPR pathways and a unique reactive oxygen species (ROS) defense in hepatocytes with high AAT aggregate load. The combination of high-sensitivity proteomics by mass spectrometry with the spatial resolution of (sc)DVP thus offers clinical insights in AATD and resilience mechanisms of affected cells, harboring the potential for tailored AATD management strategies.