Spatial single-cell mass spectrometry defines zonation of the hepatocyte proteome

Abstract

Single-cell proteomics by mass spectrometry is emerging as a powerful and unbiased method for the characterization of biological heterogeneity. So far, it has been limited to cultured cells, whereas an expansion of the method to complex tissues would greatly enhance biological insights. Here we describe single-cell Deep Visual Proteomics (scDVP), a technology that integrates high-content imaging, laser microdissection and multiplexed mass spectrometry. scDVP resolves the context-dependent, spatial proteome of murine hepatocytes at a current depth of 1,700 proteins from a cell slice. Half of the proteome was differentially regulated in a spatial manner, with protein levels changing dramatically in proximity to the central vein. We applied machine learning to proteome classes and images, which subsequently inferred the spatial proteome from imaging data alone. scDVP is applicable to healthy and diseased tissues and complements other spatial proteomics and spatial omics technologies.

Automated single-cell proteomics providing sufficient proteome depth to study complex biology beyond cell type classifications

“…Cells were counted using the Countess TM 3 FL Automated Cell Counter (SN: AMQAF2000, Invitrogen) and strained using a 5-mL cell strainer tube (SN: 352235, Falcon®) to a final concentration of 200 cells/µL for optimal cell dispensing on the cellenONE®. For the proteome perturbation experiment, HEK-293T cells were seeded in a 6-well plate (Costar® 6-well plates, 3506, Corning Incorporated) at a density of 3 x 10 5 cells/well. THP-1 cells were seeded in a T25 flask at a density of 4 x 10 5 cells/mL in Gibco RPMI media supplemented with 10% Fetal Bovine Serum, Penicillin-Streptomycin and Glutamax.…”

“…For the proteome perturbation experiment, HEK-293T cells were seeded in a 6-well plate (Costar® 6-well plates, 3506, Corning Incorporated) at a density of 3 x 10 5 cells/well. THP-1 cells were seeded in a T25 flask at a density of 4 x 10 5 cells/mL in Gibco RPMI media supplemented with 10% Fetal Bovine Serum, Penicillin-Streptomycin and Glutamax. After 8-hours, media was supplemented with LPS (final conc.…”

“…Genomics, transcriptomics and imaging methods with single-cell resolution have been shown to provide new insights to the complex cellular interplay that underlies development and disease 1–3 . Currently, most single-cell approaches aim at resolving biological heterogeneity by describing diverse sub-populations or unexpected cell types within a given sample 4,5 . In the past few years, single-cell proteomics (SCP) has developed into a viable complement to other sequencing-based omics techniques 6–11 .…”

Filter-aided expansion proteomics

“…Tissue volumes were estimated using commercially available biopsy punches based on needs (0.35 mm, 0.5 mm, 0.75 mm, 1 mm, 1.5 mm, 2 mm, 3 mm; Integra Miltex, the United States). Specifically, the formula to calculate the volume is: volume (nL) = π × (D/2F) 2 × h × 10 - 6 , where D is the diameter (µm) of the punch, F is the expansion factor calculated as the square root of the ratio of the tissue area after expansion to the area before expansion, and h is the thickness (µm) of the slice. The detection range calibration curves were estimated using Local Polynomial Regression Fitting estimation (loess) using the number of identified peptides and proteins.…”

An automated and fast sample preparation workflow for laser microdissection guided ultrasensitive proteomics

“…Follow-up work based on further improved sample preparation and LC-MS workflows recently led to the first single-cell (i.e. slice of a single cell) proteome measurements from frozen and formalin-fixed (FFPE) tissues (8, 9). In FFPE tissue, we reproducibly quantified up to 2,000 proteins from single excised hepatocyte contours (∼5,000 µm 3 in volume) based on an optimized sample preparation protocol and label-free based diaPASEF acquisition (10) on the Bruker timsTOF SCP instrument (11).…”