Background

Proteolysis is a critical cellular/biochemical process receiving extensive research attention that has found wide applications in industry and biomedicine [1–6]. Most current progress has been focused on the structures [7–9], cleavage sites [10–13], and proteolytic mechanisms of commonly seen proteases [14–17]. Meanwhile, a knowledge gap still remains regarding the molecular level details of the step-by-step, proteolytic actions on the substrate polypeptides/proteins. Bridging this gap is challenging because it requires revealing, in real time, the details of the reaction mixture, a complex ensemble containing peptide pieces with constantly changing lengths and populations. Perhaps the most feasible practice to probe the lengths and populations of the broken peptide sequences of the entire ensemble would be tandem mass spectrometry (MS/MS or MS²) [18–21], which also resolves amino acid sequence [22–24]. However, MS does not conveniently provide time-resolved information due to the change in product size and population as well as the self-degradation of the protease, which may create additional peptide sequences in the reaction mixture in real time, complicating the mass analysis and even the proteolytic kinetics due to the loss in protease over time. Thus, MS is often applied to analyze the final products after a certain stage (or upon completion) of a proteolytic reaction.

Recently, we found it possible to track the proteolytic process using site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy, which is sensitive only to the spin-labeled peptide truncations regardless of the presence of the protease and other broken peptide pieces [25]. This is because EPR line width is sensitive to the molecular size of the labeled polypeptides, which can be tracked via time-resolved, two-dimensional (2D) EPR [26–27]. We also found that immobilizing proteases on the surface of metal–organic materials (MOMs) crystals via co-crystallization allowed for the separation of protease from the soluble reaction mixture during a proteolytic reaction, which contains intact and broken peptides, by centrifugation [28–30]. The soluble portion can then be subjected to MS/MS study to determine the peptide sequences of the entire ensemble. Thus, we proposed a combination of MOMs, EPR, and MS to reveal the global (via MS) and local (via EPR) structural information of the peptide substrates in real time upon proteolysis, which ended up with a dynamic movie depicting the cleavage process [25]. As that was the first time that such a combination was applied to study proteases, we believe it is necessary to summarize the practical protocols to carry out such a complicated study. This protocol is based on our recent publication and utilized the data therein to demonstrate the protocol [25].