2.1. The isoTOP-ABPP platform for reactive-cysteine profiling

IsoTOP-ABPP is a derivative of activity-based protein profiling (ABPP), a pioneering technology for interrogating protein activity directly in complex biological systems. In general, ABPP probes contain three elements: (1) a reactive warhead for covalently labeling target proteins; (2) a reporter tag for affinity purification or fluorescence detection; and, (3) a linker to minimize steric hindrance between the reporter and reactive groups [30,31]. Early ABPP methods utilized reactive warheads targeting a specific enzyme family, such as the fluorophosphonate probe for the serine hydrolases [32]. In latter iterations, more reactive and promiscuous electrophiles were utilized [33], culminating in the use of an iodoacetamide-alkyne (IA-alkyne) probe for modification of reactive cysteines in the proteome. The isoTOP-ABPP platform couples an IA-alkyne probe with an isotopically tagged cleavable linker, enabling the selective enrichment, release, and mass-spectrometry (MS) relative quantification of IA-labeled peptides from two samples. The isoTOP-ABPP platform involves the following steps: (1) treatment of lysates with IA-alkyne to label reactive cysteines; (2) conjugation of IA-labeled cysteines in control and experimental samples to isotopically differentiated cleavable azide-biotin tags using copper-catalyzed azide-alkyne cycloaddition (CuAAC) [34]; (3) enrichment of IA-labeled proteins on streptavidin beads, followed by on-bead tryptic digestion, and linker cleavage to release IA-labeled peptides; and, (4) analysis of the resulting isotopically heavy and light peptide pairs using LC/LCMS/MS to quantify reactivity differences in two samples using light:heavy isotopic ratios [1] (Figure 1).

(A) General isoTOP-ABPP workflow. Reactive cysteine residues on two proteome samples are labeled with IA-alkyne, followed by CuAAC with an isotopically heavy or light biotin-azide cleavable linker. The two lysates are combined, biotinylated proteins are enriched on streptavidin-agarose beads, and subjected to an on-bead trypsin digestion. The IA-labeled peptides are released and analyzed by LC/LC-MS/MS. Heavy and light peptide pairs are quantified by their extracted MS1 peaks. (B) IA-alkyne structure and cysteine-labeling scheme. (C) The tobacco-etch virus (TEV) protease cleavable biotin-azide tag for isoTOP-ABPP.

Limitations in the current isoTOP-ABPP platform include the low coverage of the cellular cysteinome. Since low concentrations (100 uM) of IA-alkyne are used for proteome labeling, only 1000–2000 cellular cysteines are identified in a typical analysis. The subset of cysteine residues identified are those that demonstrate high reactivity with the IA electrophile, and have been shown to be enriched in functional cysteines [1]. However, some classes of functional cysteines remain intractable to IA labeling, and are therefore not typically captured in an isoTOP-ABPP analysis. Furthermore, reduced coverage of cysteine residues from low-abundant proteins, particular those localized within subcellular organelles, could limit the potential utility of isoTOP-ABPP for certain applications. Furthermore, cell lysates for isoTOP-ABPP are not typically treated with reducing agents, thereby limiting access to proteins that are highly susceptible to oxidation or aggregation. As discussed below, the development of new cysteine-reactive electrophiles, analytical methods, and cell-based profiling approaches, can serve to overcome some of these current limitations.