Combining Separation, Digestion, and Ionization on a Mass Spectrometry Cartridge to Enable Biomedical Research on Proteoforms

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Project Summary Each protein consists of numerous different molecular entities, termed proteoforms. These different forms arise from post-translation modifications, alternative, and genetic mutations among others. Proteoforms can have different biological function, alternations can cause disease, and they can serve as highly specific biomarkers. Proteoform analysis is, therefore, an important frontier in biomedical research. Progress is slow, however, because current technology is inadequate. On one hand, better high-performance protein measurement tools are needed for some applications to reach ever deeper into the proteoform. On the other hand, faster and simpler tools are needed for other applications to scale up proteoform research and increase participation in the field by scientists who are not protein mass spectrometry (MS) specialists. To that end, this proposal aims to develop self-contained analytical devices (‘cartridges’) that combines all of the components necessary to analyze both intact proteins and proteolytic peptides. The cartridge will include an antibody-based preconcentration system, an immobilized enzyme to perform protein digestion if desired, and a built-in substrate to perform protein ionization by electrospray. The cartridge will allow the entirety of the protein analysis workflow to occur automatically on a single device that can be directly coupled to the mass spectrometer. All of these processes will be designed to work without external pumping or other complex systems for sample handling. In Aim 1, a device will be developed that combines affinity-based protein purification with a porous material to perform electrospray ionization (substrate-supported electrospray ionization or ssESI). Proteoforms captured by the affinity material will be eluted onto the ionization material for immediate detection using top-down mass spectrometry. In Aim 2, an enzymatic digestion step will be integrated into the analysis cartridge to enable detection of proteoforms at the peptide level. While digestion of proteoforms has disadvantages, it does enable more sensitive detection and better sequencing by tandem mass spectrometry. In Aim 3, electrophoretic methods will be developed to separate and stack proteoforms on the ionization substrate. The electrophoretic manipulations will be designed to occur in minutes. Separations will be mainly designed to reduce or eliminate ion suppression, while stacking will be done to improve detection limits.