Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Novel Strategies in Top-Down Proteomics (#152)

Ying Ge 1
  1. UW-Madison, Madison, WI, United States

Top-down mass spectrometry (MS)-based proteomics is arguably the most powerful method to comprehensively characterize proteoforms that arise from genetic variations, alternative splicing, and post-translational modifications (PTMs), but myriad challenges remain. We have been developing novel strategies to address the challenges in top-down proteomics in a multi-pronged approach. To address the protein solubility challenge, we have recently identified a photo-cleavable anionic surfactant (“Azo”) that can be rapidly degraded upon UV irradiation, for top-down proteomics. Azo is MS-compatible and can effectively solubilize proteins with performance comparable to SDS. Importantly, Azo-aided top-down proteomics enables the solubilization of membrane proteins for comprehensive characterization of PTMs.  To address the proteome complexity challenge, we have been developing new chromatography materials and novel strategies for multi-dimensional liquid chromatography (MDLC) to separate intact proteins. We developed novel hydrophobic interaction chromatography (HIC) materials for high-resolution separation of intact proteins under non-denaturing mode and demonstrated the potential of online HIC/MS for top-down proteomics. Given the difficulty in detecting large proteins in top-down MS, importantly, we  developed a novel serial size exclusion chromatography (sSEC) strategy for size-based protein separation that can be coupled with online reverse phase chromatography (RPC) and high-resolution MS which enabled the top–down MS analysis of large proteins (>200 kDa). Furthermore, we established a robust top-down LC/MS-based targeted proteomics platform for quantification of protein expression and PTMs concurrently in complex mixtures with high throughput and high reproducibility.  To address the proteome dynamic range, we have been developing novel nanomaterials that can bind low abundance proteins and PTMs with high specificity. First, we designed and synthesized novel superparamagnetic nanoparticles (NPs) for capturing phosphoproteins globally out of the human proteome with high specificity. Subsequently, we developed an intergrated top-down phosphoproteomics work flow that coupled NP-based phosphoprotein enrichment by functionalized NPs with online top-down LC/MS/MS to enrich, identify, quantify, and characterize intact phosphoproteins directly from cell lysates and tissue homogenates.  To address the challenge in under-developed software, we developed MASH Suite Pro, a comprehensive software tool for top-down proteomics including protein identification, quantitation, and characterization with visual validation and versatile user-friendly interface.