Peptide Peak Areas
Joomi Ahn, senior research chemist in the biopharmaceutical sciences department at Waters, and John R. Engen, Ph.D., associate professor at Northeastern University, have been pursuing further improvements in hydrogen/deuterium exchange mass spectrometry (HXMS), specifically the degree of reproducibility of the pepsin digestion data.
HXMS involves a chemical reaction in which a covalently bonded hydrogen atom is replaced by a deuterium atom or vice versa. Usually the examined protons are the amides in the backbone of a protein, and because hydrogen exchange gives information about the solvent accessibility of various parts of the molecule, it is an important approach for generating descriptions of protein tertiary structure.
A critical feature of the procedure is the production of peptides through online acid protease digestion. By using a nonspecific protease immobilized on a column, the samples can be moved through the process train without additional isolation or purification steps.
The researchers digested a variety of proteins with pepsin and then measured the peak areas in a large-scale replicate analysis using LC/MSE methodology. The studies were performed using Waters’ nanoAcquity UPLC® and Synapt™ HDMS™ systems. The listed peptides, consistently identified more than 27 times out of 31 runs, showed an average of 6.6% relative standard deviation of the peak area, indicating a high level of reproducibility.
Peptic peptide maps resulted in extensive sequence coverage for the majority of the proteins studied, up to 100%, including in some cases the mapping of post-translational modifications and disulfide bonds by the chromatographic separation in less than 10 minutes.
“The high confidence and accuracy of peptide identification demonstrates the low variability and good reproducibility of the UPLC separation technology for analysis of these complex pepsin digestion samples,” Ahn said. “This information is highly significant for reliable conformational studies of proteins. Moreover, the rapidity of the analysis has piqued the attention of investigators using the widely applied trypsin digestion procedure,” whose adaptation for online analysis, she said, could be onerous.