Evaluation of Proteolytic Digestion Efficiency in Hydrogen Exchange–Mass Spectrometry Experiments Using the Digestible Peptide Score

In a hydrogen exchange–mass spectrometry (HX-MS)
experiment,
the enzymatic proteolysis of the deuterated protein is an essential
step. Often the differences in the performance between different digestion
protocols or between immobilized protease columns can be challenging
to evaluate. To compare differences in the performance of immobilized
protease columns, a new digestion efficiency metric known as digestible
peptide scoring (DPS) was developed and is presented in this work.
The measured response fraction of substance P peptide is used to assign
a value between 0% and 100% based on the fraction of substance P digested
by the enzyme, using angiotensin II as an undigested internal standard.
In this work, the DPS approach was tested using multiple immobilized
pepsin batches prepared using different protocols. The results demonstrate
the repeatability of DPS values for batches prepared using the same
conditions and the ability of the DPS evaluations to provide unique
values when the immobilization conditions were altered. Protein digestions
obtained with a higher scoring column were better than digestions
obtained using a lower scoring column. The DPS evaluation is simple
and quickly provides an unambiguous assessment which can be used to
evaluate an immobilized enzyme column’s suitability prior to
performing an experiment, to track performance over a column’s
lifetime, to optimize protease immobilization protocols specifically
for the quench conditions of a particular experiment, and to optimize
the digestion conditions.