Quantitative proteomics currently encompasses a variety of techniques including differential labeling of separate protein mixtures; digestion of combined, labeled protein mixtures such as cell lysates with separation of resulting peptides via multidimensional LC; peptide analysis using tandem mass spectrometry; and database searching to identify and associate peptide sequences with specific proteins as well as determination of relative protein abundance from the mass spectral data.
The historical workhorse for protein analysis has been high-resolution two-dimensional electrophoresis (2DE) combined with mass spectrometry. In this method, complex proteins are separated electrophoretically and then stained. Specific proteins are chosen for mass-spectrometric identification based on quantitative comparison of the 2DE staining patterns of control proteins. There are, however, factors limiting 2DE/mass spectrometry applicability including inability to distinguish, analyze, and quantitate low-abundance proteins. Frequently, analytical methods have required the development of reagents such as antibodies to detect specific proteins.
At the “Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industries,” which was held during CASSS’ “Mass Spec” meeting in Philadelphia last month, industry scientists described applications of mass spec to simplify and streamline protein characterization and quantitation for biotech industry applications. Presentations covered a range of topics, including an LC/MS-based assay developed to detect residual host-cell proteins in biologics and use of deconvolution to resolve isotopically unresolved multiply charged states of intact proteins.
AlphaVax has adapted LC/MS assays to quantitate the vaccines it is producing for a range of infectious diseases and cancer. The company manufactures its vaccines using an alphavirus-like replicon particle (VPR) containing an RNA that encodes a protein of interest. The VPR is used to infect a cell monolayer in culture.
Following viral particle entry into a cell, the RNA is released and translated, resulting in accumulation of the desired antigenic protein in the cell. The cells are then lysed and digested with trypsin in preparation for MS analysis. The MS portion of the assay provides a means of quantifying the resulting tryptic peptides without the need to use isotopically labeled standards.
According to Jeremy Johnson, Ph.D., senior scientist, MS-based assay development does not depend on antibody-based reagents and permits rapid assay development without specific biologically derived reagents. Quantitation of the desired protein is accomplished by measuring the total ion count of a specific tryptic peptide present in cell lysate and correlating it against a standard curve generated from the equivalent synthetic peptide standard.
Dr. Johnson explained that the MS-based assay is a platform technology that can be used to screen vaccine candidates without the need to develop a full ELISA, thereby saving considerable time and money during early-phase product development. “Quantification based on peptide total ion count requires no additional experimental steps,” he added. “The direct correlation of total ion count to peptide concentration enables peptide and, therefore, protein quantitation. This method can be developed quickly with excellent specificity and a large dynamic range.”
AlphaVax will continue to use this protein assay method in product and process development. The data collected from its use will be utilized to support ELISA development as products mature. The availability of this data for material made in early development should be useful in supporting product/process consistency throughout the development cycle, Dr. Johnson explained.