Proteases irreversibly
modify proteins by cleaving their a
mide bonds and are i
mplicated in virtually every i
mportant biological process such as i
mmunity, develop
ment and tissue repair. Accordingly, it is easy to see that deregulated proteolysis is a pathogno
mic feature of
many diseases. Most of the current infor
mation available on proteases was acquired using in vitro
methods, which reveals
molecular structure, enzy
me kinetics and active-site specificity. However, considerably less is known about the relevant biological functions and co
mbined roles of proteases in
moulding the proteo
me. Although
models using genetically
modified ani
mals are powerful, they are slow to develop, they can be difficult to interpret, and while useful, they re
main only
models of hu
man disease. Therefore, to understand how proteases acco
mplish their tasks in organis
ms and how they participate in pathology, we need to elucidate the protease degrado
me—the repertoire of proteases expressed by a cell, a tissue or an organis
m at a particular ti
me—their expression level, activation state, their biological substrates, also known as the substrate degrado
me—the repertoire of substrates for each protease—and the effect of the activity of each protease on the pathways of the syste
m under study. Achieving this goal is challenging because several proteases
might cleave the sa
me protein, and proteases also for
m pathways and interact to for
m the protease web [
Overall, C.M., Kleifeld, O., 2006. Tu
mour
microenviron
ment – opinion: validating
matrix
metalloproteinases as drug targets and anti-targets for cancer therapy. Nat. Rev. Cancer 6 (3), 227–239]. Hence, the net proteolytic potential of the degrado
me at a particular ti
me on a substrate and pathway
must also be understood. Proteo
mics offers one of the few routes to the understanding of proteolysis in co
mplex in vivo syste
ms and especially in
man where genetic
manipulations are i
mpossible.
The aim of this chapter is to review methods and tools that allow researchers to study protease biological functions using proteomics and mass spectrometry. We describe methods to assess protease expression at the messenger RNA level using DNA microarrays and at the protein level using mass spectrometry-based proteomics. We also review methods to reveal and quantify the activity state of proteases and to identify their biological substrates. The information acquired using these high throughput, high content techniques can then be interpreted with different bioinformatics approaches to reveal the effects of proteolysis on the system under study. Systems biology of the protease web—degradomics in the broadest sense—promises to reveal the functions of proteases in homeostasis and in disease states. This will indicate which proteases participate in defined pathologies and will help targeting specific proteases for disease treatments.