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.
Grappling with Sample Complexity
One of the biggest challenges in the field of proteomics is dealing with the enormous complexity found in biological samples. In a typical GeLC workflow, protein bands separated by 1-D polyacrylamide electrophoresis must first be sliced by hand and digested to liberate associated peptides into solution before analysis via mass spectrometry can be realized. In addition to being tedious, this process provides limited yield and restricts downstream analysis to tryptic peptides, rather than intact proteins.
To aid researchers in this process, Protein Discovery introduced the GELFREE™ 8100 Fractionation System. The GELFREE (Gel Elution Liquid FRaction Entrapment Electrophoresis) technology allows molecular weight fractionation using high-resolution PAGE with recovery of intact proteins in the liquid phase.
As analytes in the sample are separated, the molecular weight fractions elute from the end of the gel and are concentrated in a liquid entrapment zone. The eluted fractions are then removed with a standard pipette and are ready for downstream preparation and analysis using conventional mass spec instrumentation.
The GELFREE design features an independently controllable, eight-channel electrophoretic instrument capable of simultaneously supplying constant current or voltage to each of the eight channels in the cartridge with pre-set fraction collection steps defined through the onboard touch screen interface. All eight channels can be run in parallel and yield 15–20 fractions per channel in less than 90 minutes, according to Chuck Witkowski, president and CEO.
“The system provides highly reproducible fractionation based on molecular weight with high yield recovery in the liquid phase. Since proteins are recovered intact, valuable information such as post-translational modifications and truncations can be readily determined,” stated Witkowski.
The fractions are nonbiased, added Witkowski, who noted that recovery from GELFREE is greater than 60% across the mass range. Cartridges are provided with precision precast gels, enabling reproducibility. Up to 1 mg of total protein can be loaded on each channel, so low abundance proteins can be visualized.