Mapping proteolytic neo-N termini at the surface of living cells

Abstract

N terminomics is a powerful strategy for profiling proteolytic neo-N termini, but its application to cell surface proteolysis has been limited by the low relative abundance of plasma membrane proteins. Here we apply plasma membrane-targeted subtiligase variants (subtiligase-TM) to efficiently and specifically capture cell surface N termini in live cells. Using this approach, we sequenced 807 cell surface N termini and quantified changes in their abundance in response to stimuli that induce proteolytic remodeling of the cell surface proteome. To facilitate exploration of our datasets, we developed a web-accessible Atlas of Subtiligase-Captured Extracellular N Termini (ASCENT; http://wellslab.org/ascent). This technology will facilitate greater understanding of extracellular protease biology and reveal neo-N termini biomarkers and targets in disease.

Keywords: N terminomics; proteolysis; proteomics.

Fig. 1.

Restricting subtiligase activity to the cell surface. (A) Workflow for subtiligase N terminomics in cell lysate. Biotinylated peptide ester is represented by a green star. (B) Subcellular locations of N termini identified in a subtiligase N terminomics experiment performed using soluble subtiligase in HEK293T cell lysate. (C) Approaches for labeling cell surface N termini. (Top) Addition of soluble subtiligase to live cells. (Bottom) Expression of subtiligase fused to the PDGFRβ chain (subtiligase-TM) in cells. (D) Streptavidin-488 flow cytometry demonstrates that subtiligase-TM (red) more efficiently biotinylates cell surface N termini compared to soluble subtiligase added to cells (cyan). No enzyme control is shown in gray. (E) Streptavidin-488 flow cytometry shows that robust cell surface biotinylation is observed with activate subtiligase-TM (red) but not with the catalytically inactive C221A mutant (cyan). (F) A Western blot of subtiligase-TM-expressing HEK293T cells shows that biotinylation activity is dependent on both active subtiligase and the presence of a biotinylated peptide ester substrate. No biotinylation is observed when the inactive subtiligase-C221A-TM mutant is used or in the absence of biotinylated peptide ester substrate. (G) Fluorescence microscopy shows that subtiligase-TM expression (red) and biotinylation activity (green) are colocalized at the cell surface. No biotinylation activity is observed when the inactivate subtiligase-C221A-TM mutant is expressed.

Fig. 2.

Cell surface N terminomics with subtiligase-TM. (A) Workflow for cell surface N terminomics with subtiligase-TM. (B) Subcellular locations of N termini identified in a subtiligase-TM N terminomics experiment performed in HEK293T cells. (C) Topological locations of N-terminal peptides identified in the subtiligase-TM N terminomics experiment (Top) or a subtiligase lysate experiment (Bottom). (D) Distribution of membrane protein types observed in the subtiligase-TM N terminomics experiment. (E) Distribution of distances between cleavage sites identified in the subtiligase N terminomics experiments and the corresponding transmembrane domains.

Fig. 3.

Comparison of subtiligase-TM to subtiligase-TM mutants and to other N terminomics methods. (A–D) iceLogos for the N-terminal sequence specificity of subtiligase added to cell lysate (A), wild-type subtiligase-TM (B), subtiligase-Y217K-TM (C), and subtiligase-Y217D-TM (D). (E) Fraction of subtiligase-captured N termini observed in TopFINDer datasets (gray) or not observed in TopFINDer datasets (blue) for soluble subtiligase (Left) or subtiligase-TM (Right). (F) Evidence for subtiligase-TM–captured N termini that were previously observed in TopFINDer datasets.

Fig. 4.

Quantitative subtiligase-TM N terminomics in pervanadate-treated vs. untreated cells. (A) Pervanadate treatment leads to proteolytic shedding of cell surface proteins. (B) Western blot showing an accumulation of phosphotyrosine on pervanadate treatment of cells. (C) Volcano plot showing the log₂-fold change of N termini measured by quantitative mass spectrometry in pervanadate-treated vs. untreated cells. The significance of the changes is indicated by the −log₁₀ P value (y-axis). (D) Flow cytometry validation of changes in the N-terminal proteome observed in the mass spectrometry dataset. Monoclonal antibodies that bind epitopes N-terminal to the observed cleavage site were chosen such that a decrease in signal on pervanadate treatment was expected.