Nanomechanical in situ monitoring of proteolysis of peptide by Cathepsin B

Abstract

Characterization and control of proteolysis of peptides by specific cellular protease is a priori requisite for effective drug discovery. Here, we report the nanomechanical, in situ monitoring of proteolysis of peptide chain attributed to protease (Cathepsin B) by using a resonant nanomechanical microcantilever immersed in a liquid. Specifically, the detection is based on measurement of resonant frequency shift arising from proteolysis of peptides (leading to decrease of cantilever's overall mass, and consequently, increases in the resonance). It is shown that resonant microcantilever enables the quantification of proteolysis efficacy with respect to protease concentration. Remarkably, the nanomechanical, in situ monitoring of proteolysis allows us to gain insight into the kinetics of proteolysis of peptides, which is well depicted by Langmuir kinetic model. This implies that nanomechanical biosensor enables the characterization of specific cellular protease such as its kinetics.

Figure 1. Schematic illustration of nanomechanical, in situ monitoring of proteolysis of peptide chains on the surface by using resonant microcantilever immersed in a liquid.

(A) A microcantilever was functionalized by peptide chains (PEGylated GFLG) through aminated cantilever surface. The fundamental resonance of a cantilever is given by ω = (K/M_c)^1/2, where K is the overall stiffness of a cantilever, and M_c is the overall mass of a cantilever. (B) Chemical structure of PEGylated GFLG (GlyPheLysGly) chains on a cantilever and proteolyzed peptides by protease (i.e. PEG-GF and LG sequence immobilized on a cantilever). (C) When GFLG peptides immobilized on a cantilever was exposed to protease (CTSB) in acidic medium, catalytic Cys25 and His159 of CTSB induces the successful cystein protease of GFLG, leading to proteolysis of GFLG. Such proteolysis phenomenon reduces the overall mass of a cantilever, and consequently, the increase of the fundamental resonance.

Figure 2. Relationship between proteolysis efficiency, r, and CTSB (Cathepsin B) concentration, [CTSB] is shown.

Here, proteolysis efficiency, r, is defined as r = Δm_P/Δm_I, where Δm_P and Δm_I represent the total mass of cleft peptides and immobilized GFLG peptides on the cantilever surface, respectively. The mass of immobilized peptides is measured from the resonance frequency difference between a bare cantilever and cantilever functionalized by peptides, while the mass of cleft peptides driven by protease is evaluated from the resonance difference between cantilever functionalized by peptides and such a cantilever in exposure to CTSB. The proteolysis efficiency is exponentially proportional to the CTSB concentration in buffer solution. Here, the measurement of resonant frequency shifts due to proteolysis based on 4 different cantilevers (e.g. Table 1) was implemented in dry air.

Figure 3. In situ resonant frequency shifts, which are measured in buffer solution, attributed to proteolysis of tetrapeptide GFLG are shown with three different CTSB concentrations; (A) [CTSB] = 0.28 µM, (B) [CTSB] = 0.56 µM, and (C) [CTSB] = 0.84 µM.

Further, the mass of cleft peptides driven by protease (with a given protease concentration) is computed from in situ resonant frequency shift measured in buffer solution due to proteolysis. These resonant frequency shifts with respect to time are well fitted with the Langmuir kinetic model, which allows for extraction of rate constant for proteolysis, k_p. (D) The rate constant for proteolysis, k_p, extracted from in situ resonant frequency shift is shown to be linearly proportional to CTSB concentrations, [CTSB]. This indicates that the proteolysis can be controlled by enzyme concentrations, which will be related to drug design.