Picoelectrospray Ionization Mass Spectrometry for Top-down Proteomics

Project Summary/Abstract The proteome reflects the physiology and pathology states of a patient therefore proteomics is a powerful tool for early diagnostics of diseases and monitoring of therapeutic responses. Mass spectrometry (MS) measures the mass-to-charge ratio of charged species and has become the enabling technology for proteomics. However, majority of the current proteomics studies rely on bottom-up approaches. In this case, mixtures of proteins are digested by one of the proteases (e.g., trypsin), separated by liquid chromatography (LC), and analyzed by electrospray mass spectrometry (ESI-MS). Despite tremendous successes, there remain two major limitations in bottom-up proteomics: first, it is difficult to identify all protein isoforms or proteoforms, including splicing, modifications, cleavages, etc.; second, the native state of proteins is always lost after digestion. There is currently a great push to implement top-down proteomics, i.e., identification and characterization of full-length proteins by LC-MS. Unfortunately, top-down proteomics proves to be much more challenging. There are several bottlenecks: first, lower MS sensitivity of proteins relative to peptides; second, limitation on detection of high molecular weight proteins; third, inefficient identification of proteins by MS/MS fragmentation; and fourth, laborious multidimensional protein separation not suitable for small volumes of biological samples. The field is calling for transformative technologies. In this Phase II project, Newomics Inc. proposes to further develop and commercialize a new technology, nanoflow LC-picoelectrospray ionization mass spectrometry (NanoLC- PicoESI-MS), for top-down proteomics of small-volume biological samples down to single cells. The technology is built on our microfabricated monolithic multinozzle emitters (M3 emitters) and multinozzle emitter array (MEA) chips for LC-nanoESI-MS, which collectively offer a straightforward yet novel solution to the longstanding problem of the efficient coupling between silicon microfluidic chips and ESI-MS, and pave the way for the large- scale integration on the proposed microfluidic chips for nanoLC-picoESI-MS. Our new PicoESI-MS platform will directly address the aforementioned bottlenecks, and thus enable high-sensitivity, high-throughput, and multiplex top-down proteomics of small volumes of biological samples, thereby contributing to precision medicine.