Background

The advances in next-generation sequencing and long-read sequencing have opened the floodgate of many complex genomes. Inferring protein functions based on genome annotation may misannotate the catalytic properties to the uncharacterized genes, and it must be experimentally validated (Gerlt, 2016). Studying global analysis of gene transcription and translational changes by genomic and proteomic approaches gives only partial and indirect information about protein function. Further, protein activities are subjected to various post-translational modifications and regulations, which have pronounced effects on protein function (Bürkle, 2001). Therefore, conventional proteomic or genomic methods provide only an indirect assessment of the functional state of the enzyme. The lack of effective small-molecule probes for functional characterization has hindered our understanding of a myriad of functions and regulation modes. Activity-based proteomics is an analytical platform ideally suited for the global profiling of protein function (Schmidinger et al., 2006). In this approach, the functional annotation of uncharacterized proteins can be done precisely in an activity-dependent manner. Activity-based protein profiling (ABPP), pioneered by Cravatt and Bogyo, emerged as an efficient chemical proteomic approach for the analysis of functional states of target proteins in native biological systems (Evans and Cravatt, 2006, Cravatt et al., 2008, Niphakis and Cravatt, 2014). The application of ABPP depends on the quality of the probe, a small molecule that covalently binds to the active site of the target protein. In principle, an ABPP probe contains three parts: i) the reactive group, that forms a covalent bond with a conserved active site nucleophile, ii) a linker region, that attaches the reactive group with a reporter tag, and iii) a reporter tag, that provides a handle to detect and measure the protein labelled by the reactive group.

Since its inception, the most mature format for the visualization and characterization of probe-labelled proteomes in ABPP experiments is the in-gel fluorescence scanning (IGFS) (Patricelli et al., 2001). The identity of the proteins detected in IGFS can be achieved by enrichment followed by classical in-gel trypsin digestion (Dolui and Vijayaraj, 2020a; Latha et al., 2021). For example, replacement of a fluorescence tag with desthiobiotin facilitates the enrichment of the labelled enzymes via streptavidin/avidin affinity purification and subsequent identification through the mass spectrometric platform (Cravatt et al., 2008). This gel-based ABPP method directly links the labelling patterns of in-gel fluorescence signals and their identities (Phillips and Bogyo, 2005). However, there are some limitations to the gel-based ABPP method. The major limitation is the poor resolution of proteins that co-migrate on the gel, and identifying low-abundance proteins is difficult. With the advancement in proteomic techniques, ABPP has also significantly progressed from a gel-based to a virtually gel-free mode (Park et al., 2012). Since the activity-based probe covalently binds to the active site of functional proteins, the identity of the protein with the precise active-site residue information can be determined.

In this study, we described a gel-free format of ABPP for serine hydrolases active-site peptide profiling, using an active-site targeted probe (Dolui and Vijayaraj, 2020a). It provides comprehensive information for the active serine hydrolases with specific active-site residues mapping. In this approach, the number of probe-labelled active site peptides (in the trypsin digest of a probe-labelled proteome) logically equals the number of labelled proteins present in the sample (Okerberg et al., 2005). This gel-free ABPP format offers many advantages. First, performing an affinity enrichment step before mass spectrometry analysis helps to identify a low copy number of target enzymes. Therefore, it is high throughput in nature. Second, every single unique peptide will be able to reveal the identity of the parent target proteins. Since this peptide carries active site information, it is advantageous, and this information can be utilized for designing inhibitors for those target proteins. However, one of the prerequisites of this method is the accessibility for the active sites of enzymes in a native biological system. Therefore, pre-clearing the sample is an inevitable step prior to active site peptide profiling. Hence, we have developed a single-step pre-clearing method for lipid-rich samples for serine hydrolase labelling (Dolui and Vijayaraj, 2020b). Finally, multiple controls along with unbiased selection criteria should be adopted, to rule out any background and nonspecific proteins.