Nano LC/MS Ultratrace Proteomic Analysis Using Narrow Bore Porous Layer Open Tubular Based Platform

Liquid chromatography coupled to a mass spectrometer as detector (LC-MS) are important tools for modern sample analysis especially in protein characterization and complex biological sample profiling. One specific challenge in medical analysis is that for many human or animal samples, the total amount of sample that can be used for LC-MS analysis is generally very limited. As an example, circulating tumor cells (CTCs) are usually found in blood in the level of 10 cells/mL. Despite the limited availability of CTCs, they can be useful for early therapeutic diagnosis as well as leading to effective treatment by targeting the key components in certain diseases. As the need arises for more sensitive and more efficient analysis for complex bio-medical substances, separation and analytical science are rapidly developing. Among the advances, porous layer open tubular (PLOT) columns have demonstrated high sensitivity for the analysis of complex biological samples and good adaptability to the state-of-art nanoliter-scale separation hardware without the need of extremely high backpressure, making them ideal for ultratrace clinical sample analysis. The purpose of this dissertation is to develop a practical PLOT column-based preparation, separation and analysis for limited numbers of cells as low as few hundred and also to push the limit towards extreme trace amounts of sample with even lower cell numbers.;In Chapter 1, a step by step technology review is presented beginning with the different techniques of limited sample collection to the sample preparation methods. Nano LC gradient separation and related types of columns are discussed, followed by the introduction to modern mass spectrometry and deep proteomic profiling software and strategies.;In Chapter 2, polystyrene-divinylbenzene (PS-DVB) based PLOT columns and monolithic SPE columns were prepared. A proteomics separation platform was assembled and tested utilizing the PLOT column coupled with a Q-Exactive mass spectrometer demonstrating the potential of deep proteomic profiling for complex biological samples. Theoretical comparisons have been performed between the nanoflow PLOT column and conventional packed columns at normal flow rate. Furthermore, modifications have been made to the PLOT column and monolithic SPE column to further improve their hydrophobicity and separation efficiency.;In Chapter 3, Adaptive Focused Acoustics (AFA) sonication technology and magnet-activated cell sorting methods were integrated to the previously assembled SPE-PLOT proteomics separation platform. Limited amounts of MCF-7 cells spiked into human blood were specifically captured and processed before separation and profiling. System sensitivity as low as 10 zmol was achieved in the parallel reaction monitoring mode as well as a 4--5 fold improvement in the proteomics profiling depth for the limited cell analysis using 20 nL/min nanoflow on a high resolution/accurate mass (HR/AM) Q-Exactive mass spectrometer.;In Chapter 4, considering the sensitivity that the PLOT column can reach against the total number of cells used for profiling in the previous tests, a microreactor based sample processing method was employed as part of the investigation of even lower amount of bio-medical sample preparation and analysis using both online and offline approaches. AML cells and mouse brain motor neurons were selected as the test samples for method development and validation. Preliminary proteomics data was acquired and future research directions were discussed to further validate the method.