Multiplexed Probing of Proteolytic Enzymes Using Mass Cytometry-Compatible Activity-Based Probes

Abstract

The activome can be considered as a subset of the proteome that contains enzymes in their catalytically active form and can be interrogated by using probes targeted towards individual specific enzymes. A subset of such enzymes are proteases that are frequently studied with activitybased probes, small inhibitors equipped with a detectable tag, commonly a fluorophore. Due to the spectral overlap of these commonly used fluorophores, simultaneous analysis becomes limited.To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry. Using lanthanide-based tags instead of fluorophores gives us the ability to monitor the activity of multiple proteases in parallel. As proof of concept we developed a panel of cathepsin and legumain specific probes and showed that we were able to identify an activome of these proteases in two cell lines and peripheral blood mononuclear cells, providing a framework for the use of mass cytometry for multiplexed enzyme activity detection.

Activity-Based Probes to Utilize the Proteolytic Activity of Cathepsin G in Biological Samples

“…CyTOF (Gartner et al, 2020;Poreba et al, 2020) offers an advanced tool for deep profiling of cells for their extracellular as well as intracellular proteolytic activity of proteases and increases the analytical and mechanistical resolution of the method even further.…”

“…Remarkably, metal-tagged, time-of-flight activity-based probes (also called TOF probes) were generated by incorporating an N-terminal tetracarboxylic acid (DOTA)-chelated stable isotope of lanthanoids with a C-terminal acyloxymethylketone (AOMK) attached to the diphenyl phosphonate to simultaneously detect four different protases, CatB, CatL, asparagine-endoprotease (AEP also called legumain), and neutrophil elastase, in cell lines and PBMCs by using CyTOF as well as imaging mass cytometry (IMC) ( Poreba et al, 2020 ). TOF probes, 159 Tb CatB, 175 Lu CatL, 158 Gd legumain, and 159 Tb NE, were applied to detect CatB, CatL, legumain, and NE in immune cells using CyTOF, and high legumain was found in B cells of one donor indicating infection or cancer ( Poreba et al, 2020 ). Indeed, the proteolytic content of legumain (AEP) was compared between primary human B cells isolated from PBMCs (primary CD22 + B cells) and the EBV-transformed B cell line and found high levels of AEP in the EBV-transformed B cell line in contrast to primary CD22 + B cells.…”

“…It is unlikely that CyTOF will replace regular flow cytometry due to the destruction of the cells during measurement (it is not possible to sort cells) and the reduced flow rate ( Gadalla et al, 2019 ). The opportunity to add ABPs to CyTOF ( Gartner et al, 2020 ; Poreba et al, 2020 ) offers an advanced tool for deep profiling of cells for their extracellular as well as intracellular proteolytic activity of proteases and increases the analytical and mechanistical resolution of the method even further.…”

“…The overexpression of CatG in glioblastoma cells was demonstrated to downregulate cell surface MHC I molecules, and CatG activity was verified by using the MARS116 probe ( Palesch et al, 2016 ). NSPs, including CatG, are unevenly located in the granules of neutrophils ( Kasperkiewicz et al, 2017 ), and neutrophil elastase can be detected in various immune cells by CyTOF ( Poreba et al, 2020 ). On the right panel, CatG-specific ABPs could be used to investigate protease activity in various physiopathological conditions such as photoaged human skin, cancer, Papillon–Lefevre syndrome, inflammation, and infection [ Hu et al, 2020 ; Korkmaz et al, 2010 ; Burster et al, 2020 )].…”

“…This would meet the effort to gain mechanistic insights even with a limited clinical material and would fit well to a standardized CyTOF workflow for system-level biomarker discovery. Moreover, the principle of assessing the proteolytic activity of CatG by using a biotin-coupled ABP to detect proteolytic activity at the cell surface ( Gartner et al, 2020 ) or more advanced by using ABPs with N-terminal tetracarboxylic acid-chelated stable isotope of lanthanoids ( Poreba et al, 2020 ) could be applied to the progressive field of CyTOF and optical imaging by combining multidimensional single-cell analysis with histological stratification. An example of this combination is the Hyperion imaging mass cytometry technology that offers spatially resolved single-cell analysis using mass tagged antibodies ( Jackson et al, 2020 ).…”