Degradomics
Degradomics is a sub-discipline of biology encompassing all the genomic and proteomic approaches devoted to the study of proteases, their inhibitors, and their substrates on a system-wide scale.
As the second largest class of enzymes behind ubiquitin ligases and responsible for ~2% of any organism's genes,[4] proteases have drawn the attention of biologists to develop a field aimed at identifying and quantifying their roles in biology.
These bioactive molecules play roles in coagulation, complement activation, DNA replication, cell-cycle control, cellular proliferation and migration, hemostasis, immunity, and apoptosis.
[5][6] The degradome was broken down into two concepts, the first referring the entire profile of proteases expressed under by a cell, tissue, or organism under defined circumstances.
Extracted total RNA serves as a template for complementary DNA (cDNA) that is tagged with fluorescent probes before being allowed to hybridize to the microarray for visualization.
[2] A more sensitive approach to transcript analysis of a gene is quantitative real-time polymerase chain reaction (qRT-PCR), which has also seen action in quantifying protease mRNA levels.
While developed microarrays remain a major workhorse in studying gene expression in degradomics, its limitations of cross hybridization and dynamic range issues suggest RNA-seq will take a larger role as costs decrease and analysis improves.
While useful in early degradomic studies, the limitations of adapted yeast two-hybrid screens have forced the field to move on to higher-throughput approaches for protease-substrate discovery.
Unfortunately, these assays fail to provide insight on enzymatic function for proteases and suffer similar drawbacks to western blots regarding reliable quantification.
Unfortunately, in addition to providing little functional information, IHC is also non-quantitative, making it an unappealing option for describing degradomics on system-wide scales.
Two-dimensional Polyacrylamide Electrophoresis (2D-PAGE) gels historically compared intensities from protease treated and untreated sample spots in order to identify possible candidate substrates.
Conventional shotgun proteomics identification of low abundance proteins in samples remains limited despite advances in Mass Spectrometry (MS) technology.
These techniques have coalesced into a new field of positional proteomics or terminomics aimed at identifying protein N- or C-terminal modifications of protease substrates.
[8] Terminomic approaches including Terminal Amine Isotopic Labeling of Substrates (TAILS) N-Terminomics, Combined FRActional Diagonal Chromatography (COFRADIC), and C-Terminomics add the level of stringency to conventional shotgun proteomics necessary to make them workhorse of degradomics.
The polymer ignores the unreactive primary amines blocked by their tags, allowing them to be separated from trypsin generated peptides by ultrafiltration for Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) analysis.
Recently, the Overall Lab tackled another difficulty of C-terminomics, using endopeptidase LysargiNase™ to generate C-termini carrying N-terminal lysine or arginine residues.
[40] Beginning with a peptide library generated from endopeptidase digestion of a proteome, this technique allows for screening and characterizing the prime- and non-prime specificity for proteases.
Now, protease generated primary amines that constitute the prime site of cleavage can be biotinylated and isolated due to their reactivity and analyzed by LC-MS/MS.
Limitations including difficult production, specificity, stability, and toxicity[23] hamper ABP development but these probes have proved useful in revealing protease biological activity and remain a promising avenue in degradomic technology.
[45][46][47] PSPs do not depend on targeting active proteases with tagged compounds but rather on quantitative proteomics using stable isotope labeled standard peptides.
This is thanks to trypsin treatment for mass spectrometry generating peptides specific to inactive zymogen precursors, active proteases, or common to both forms.
Experiments using iTRAQ labeling and LC-MS/MS with STEP and PSP peptide internal standards have successfully quantified total and active protease levels in biological samples.
[23] Experiments using iTRAQ labeling and LC-MS/MS with STEP and PSP peptide internal standards have successfully quantified total and active protease levels in biological samples.
Owing to the increasing complexity of regulation of cellular processes and the roles proteases play in them, bioinformatics continues to be an invaluable tool for degradomics.
[55][56] Proteolytic processed N-termini have been proposed as potential biomarkers[57] as disease specific proteolysis has been well studied in pathologies such as inflammation [58] and cancer.
[61] Advancements in SRM and MRM clinical assays also allow for analyzing proteolytic signature biomarkers in patient samples and can be complemented by PSP quantification.
