Single cell analysis such as single cell sequencing has shed invaluable light on cellular heterogeneity and understanding molecular mechanisms such as cell differentiation. Modern single cell sequencing techniques have achieved throughput of analyzing thousands of single cells per day with sequencing depth of thousands of genes. Proteins play vital roles in almost all biological processes and have been crucial biomarkers for disease diagnosis and drug development. Unlike DNA and RNA molecules, protein molecules can’t be amplified, making the large-scale characterization of proteins (proteomics) challenging for mass-limited samples such as single cells. Novel proteomics methodologies with extremely high sensitivity are vital for analysis of mass-limited samples. This work focuses on developing ultrasensitive Mass Spectrometry (MS)-based proteomics platforms to enable large-scale proteome profiling of mass-limited samples. In Chapter 2, we applied nanoRPLC-CZE-MS/MS platform for large scale proteome profiling on 5 μg of a MCF7 cell digest. The digest was fractionated by the nanoRPLC, followed by dynamic pH junction based CZE-MS/MS. The nanoRPLC-CZE-MS/MS produced over 7500 protein IDs and nearly 60000 peptide IDs from the 5-μg MCF7 proteome digest. It reduced the required amount of complex proteome digests for LC-CZE-MS/MS-based deep bottom-up proteomics by two orders of magnitude. In Chapter 3, we improved the sensitivity of the nanoRPLC-CZE-MS/MS system drastically. The improved system identified 6500 proteins from a MCF7 proteome digest starting with only 500-ng peptides using a Q-Exactive HF mass spectrometer. In addition, we coupled single spot solid phase sample preparation (SP3) method for sample preparation on 5000 HEK293T cells, resulting in 3689 protein IDs with the consumption of a peptide amount that corresponded to only roughly 1000 cells. In Chapter 4, we developed a Nanoparticle-aided Nanoreactor for Nanoproteomics (Nano3) technique for processing few mammalian cells for bottom-up proteomics. The Nano3 technique employed nanoparticles packed in a capillary channel to form a nanoreactor (≤30 nL) for concentrating, cleaning, and digesting proteins followed by nanoRPLC-MS/MS analysis. The Nano3 method identified 40-times higher number of proteins from 2-ng mouse brain protein samples compared to the low volume SP3 method. The Nano3 method was further applied in processing 10-1000 HeLa cells for bottom-up proteomics, producing 1084 ± 287 (N=4) protein IDs from only 10 HeLa cells using a Q-Exactive HF mass spectrometer. In Chapter 5, we developed a universal sample preparation method for denaturing top-down proteomics (dTDP), and the method combined the sodium dodecyl sulfate (SDS)-based protein extraction and the membrane ultrafiltration (MU)-based protein cleanup. The MU method outperformed CMP (chloroform-methanol precipitation) and SP3 methods, resulting in high and reproducible protein recovery from both E. coli cell (59±3%) and human HepG2 cell (86±5%) samples without a significant bias. The assay afforded identification of various post-translational modifications and protein containing transmembrane domains through top-down analysis.
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