Next Generation Mass Spectrometry Data Acquisitions for Low-input to Single-Cell Proteomics.

Abstract

Single-cell proteomics provides an excellent opportunity to understand cellular heterogeneity and design functional experiments to test the role of individual cells in complex biochemical systems. However, profiling proteins from individual cells presents many analytical challenges, including how to process single-cell samples, detect trace peptide ion signals, quantify proteins accurately, and improve analysis throughput to measure large numbers of cells within a reasonable timeframe to draw meaningful biological conclusions. This work presents the development of novel strategies to address these analytical needs using capillary electrophoresis-mass spectrometry (CE-MS) for studying single-cell heterogeneity and its formation in live Xenopus laevis (frog) embryos. Specifically, to improve detection sensitivity, three novel data acquisition techniques, termed electrophoresis correlative mass spectrometry (Eco-MS), were developed for use with both Orbitrap and timsTOF Pro mass spectrometers. By efficiently utilizing the limited duty cycle of the mass spectrometer, Eco-MS was able to detect 4.5-times more proteins than the conventional data acquisition when analyzing single-cell equivalent proteome digests. Additionally, an ultra-fast CE-MS workflow was developed, requiring only a 15-minute effective separation window to profile over 1,000 proteins from the subcellular proteome. Using the ultra-sensitive and high-throughput CE-MS platforms developed in this dissertation, single-cell proteome heterogeneity was uncovered in early-blastula stage Xenopus embryos. To understand the formation of single-cell proteome heterogeneity, the subcellular proteome contents were analyzed before asymmetric cell division. The knowledge gained from this study provided valuable insights into the possible mechanisms of asymmetric cell division, a fundamental process for generating heterogeneous cells during embryonic development.