Deconvolution of Isotopically Unresolved Intact Proteins
For spectra acquired through electrospray ionization of LC-separated biomolecules, charge deconvolution to yield enhanced ion statistics by combining peak intensities for all detected charge states is generally required. Maximum entropy charge deconvolution can produce spectra characterized by higher resolution and correct isotope distribution, enabling accurate determination of molecular weights and quantitation.
At the symposium, Natalia Belyaeva, Ph.D., a scientist at Thermo Fisher Scientific, described an alternative approach for resolving multiply charged proteins using a method based on graph search of all optimal paths.
Dr. Belyaeva explained that existing algorithms may not allow processing of data because spectral line widths vary with m/z region, even for the same compound. As a result, she and her coworkers developed an algorithm to resolve multiply charged protein entities utilizing a graph search of all optimal paths. All charge states that can belong to the compound form an individual path on the graph consisting of all determined peaks. They then applied the algorithm to analyzing different modifications such as glycosylation of an IgG.
“This algorithm assigns a score based on each determined charge state chain based on m/z values, peak quality, and charge envelope shape,” Dr. Belyaeva explained. The score assigned to each potential charge-state chain facilitates choosing the “best hypothesis” for all possible charge-state chains, and can avoid assigning a charge state to wrong mass.
To increase data quality by decreasing random noise and improving the charge-state envelope, scientists performed optimal averaging of spectra before the deconvolution algorithm was applied. The scientists showed that a deconvolved spectrum, after appropriate averaging to “get enough charge states and improve the charge-state envelope” revealed multiple glycosylation sites, as well as several antibody variants.
“High-accuracy deconvolution provides not only correct protein molecular weight determination, but also accurate quantification, which is important for such areas as characterization of different protein modifications or drug conjugates and can be applied to data acquired using both orbitrap and ion traps,” Dr. Belyaeva said.
All of these novel applications are expected to help biotechnologists identify, characterize, and quantitate complex protein therapeutics produced in a variety of recombinant systems.