Mapping Cell Surface Proteolysis with Plasma Membrane-Targeted Subtiligase

Abstract

N terminomics methods combine selective isolation of protein N-terminal peptides with mass spectrometry (MS)-based proteomics for global profiling of proteolytic cleavage sites. However, traditional N terminomics workflows require cell lysis before N-terminal enrichment and provide poor coverage of N termini derived from cell surface proteins. Here, we describe application of subtiligase-TM, a plasma membrane-targeted peptide ligase, for selective biotinylation of cell surface N termini, enabling their enrichment and analysis by liquid chromatography-tandem MS (LC-MS/MS). This method provides increased coverage of and specificity for cell surface N termini and is compatible with existing quantitative LC-MS/MS workflows.

Keywords: Cell surface; Enrichment; Mass spectrometry; N terminomics; Post-translational modification; Proteolysis; Proteomics.

Figure 1.. Subtiligase-TM workflow.

A stable cell line expressing subtiligase-TM is treated with a biotinylated subtiligase substrate (TEV Ester 6) to effect biotinylation of cell surface N termini. After labeling, the cells are lysed, biotinylated proteins are enriched on neutravidin resin, and a trypsin digestion is performed to remove internal peptides. N-terminal peptides are then selectively eluted by cleavage of a TEV protease site found in the subtiligase substrate and are analyzed by LC-MS/MS.

Figure 2.. Structure of TEV Ester 6 (TE6).

TE6 is a peptide ester with the sequence biotin-EEENLYFQ-⍺-aminobutyric acid-glycolate ester-R. The peptide is typically synthesized as a C-terminal amide.

Figure 3.. Fluorescence images of HEK293T cells stably expressing subtiligase-TM.

Subtiligase-TM (SL-TM) expression is detected with an anti-FLAG antibody conjugated to Alexa Fluor 647 and biotinylation activity is detected with streptavidin conjugated to Alexa Fluor 488.