Assessing Accuracy of an Analytical Method in silico: Application to “Accurate Constant via Transient Incomplete Separation” (ACTIS)
2020
Analytical
methods may not have reference standards required for testing their
accuracy. We postulate that accuracy of an analytical method can be
assessed in the absence of reference standards in
silico
if the method is built upon deterministic processes. A deterministic
process can be precisely computer-simulated thus allowing virtual
experiments with virtual reference standards. Here, we apply this in
silico
approach to study “Accurate Constant via
Transient Incomplete Separation” (ACTIS), a method for finding the
equilibrium dissociation constant (Kd)
of protein–small molecule complexes. ACTIS is based on a
deterministic process: molecular diffusion of the interacting
protein–small molecule pair in a laminar pipe flow. We used COMSOL
software to construct a virtual ACTIS setup with a fluidic system
mimicking that of a physical ACTIS instrument. Virtual ACTIS
experiments performed with virtual samples — mixtures of a protein
and a small molecule with defined rate constants and, thus, Kd
of their interaction — allowed us to assess ACTIS accuracy by
comparing the determined Kd
value to the input Kd
value. Further, the influence of multiple system parameters on ACTIS
accuracy was investigated. Within multi-fold ranges of parameters,
the values of Kd
did not deviate from the input Kd
values by more than a factor of 1.25 strongly suggesting that ACTIS
is intrinsically accurate and that its accuracy is robust.
Accordingly, further development of ACTIS can focus on achieving high
reproducibility and precision. We foresee that in
silico
accuracy assessment, demonstrated here with ACTIS, will be applicable
to other analytical methods built upon deterministic processes.
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