Proteomics, the intricate study of the proteome, has evolved significantly with advancements in mass spectrometry (MS)-based techniques. While bottom-up proteomics (BUP), relying on proteolytic peptides, offers extensive protein identification, it grapples with challenges like limited sequence coverage and ambiguity in proteoform differentiation. Conversely, top-down proteomics (TDP), targeting intact proteoforms, provides a comprehensive view, capturing post-translational modifications (PTMs) but is constrained by low sensitivity and complex fragmentation. Analytical techniques like capillary zone electrophoresis (CZE) and liquid chromatography (LC) play pivotal roles in enhancing separation efficiency, while ion mobility spectrometry (IMS) complements mass spectrometry by offering an additional dimension of gas-phase separation. The unity of multi-dimensional separations and cutting-edge bioinformatics tools has expanded the horizons of proteomics, yet challenges remain. In chapter 2, we engineered a high-throughput BUP workflow for plasma and serum analysis by integrating nanoparticle (NP) protein corona formation, rapid on-bead tryptic digestion, and CZE-tandem mass spectrometry (CZE-MS/MS). Four distinct magnetic NPs with varied functional groups on their surfaces using SDS-PAGE and CZE-MS/MS on healthy human plasma were firstly evaluated. The optimized workflow with amine-terminated and carboxylate-terminated NPs to analyze serum samples from both healthy and NUT cancer-afflicted mice was applied. This approach facilitated the identification of hundreds of proteins from plasma and serum samples, achieving high throughput within only 3.5-hours from sample to data. Leveraging the NP protein corona, rapid digestion, and CZE-MS/MS, we unveiled potential cancer biomarkers through quantitative proteomics. In chapter 3, we explored magnetic NP-based immobilized metal affinity chromatography (IMAC) using Ti4+ and Fe3+ for the selective enrichment of phosphoproteoforms from a standard protein mixture and yeast cell lysate. This method demonstrated reproducible, high-efficiency enrichment, outperforming a commercial phosphoprotein enrichment kit in terms of capture efficiency and recovery. Reversed-phase LC (RPLC)-MS/MS analysis of yeast cell lysates post-IMAC enrichment yielded nearly 100% more phosphoproteoform identifications than without enrichment. Intriguingly, phosphoproteoforms identified after Ti4+-IMAC or Fe3+-IMAC enrichment corresponded to proteins of significantly lower abundance and distinct pools, suggesting their combination could enhance phosphoproteome coverage. These findings underscore the potential of our magnetic NP-based Ti4+-IMAC and Fe3+-IMAC in advancing top-down MS characterization of phosphoproteoforms in complex biological systems. In chapter 4, we introduced the first integration of CZE, IMS, and MS for online multi-dimensional separations of histone proteoforms. This innovative CZE-high-field asymmetric waveform IMS (FAIMS)-MS/MS platform enabled the identification of 366 histone proteoforms (ProSight PD) and 602 (TopPIC) from a commercial calf histone sample, using only a low microgram starting material. Remarkably, CZE-FAIMS-MS/MS achieved a threefold increase in histone proteoform identifications compared to CZE-MS/MS alone. These findings suggested that CZE-FAIMS-MS/MS holds significant potential for the comprehensive and highly sensitive characterization of histone proteoforms, paving the way for deeper insights into their complex roles in epigenetic control. In chapter 5, we advanced native proteomics by analyzing large proteoforms and protein complexes, up to 400 kDa, from complex proteomes using native CZE (nCZE) coupled with an ultra-high mass range (UHMR) Orbitrap mass spectrometer. Our nCZE-MS technique successfully measured a 115-kDa standard protein complex with minimal sample consumption of only 0.1 ng. When applied to an E. coli cell lysate, nCZE-MS detected 72 proteoforms and complexes in the 30-400 kDa range from only 50 ng of material in a single run. Notably, the mass distribution of detected proteoforms and complexes was consistent with mass photometry measurements, marking a technical leap in native proteomics for complex proteome analysis.
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