Development of an Analytical Platform for Quantitative Measurement of Biomolecular Dynamics

Abstract

Chapter 1: High-throughput Measurements of Lipid Turnover Rates Using Partial Metabolic Heavy Water Labeling Novel analytical platforms for high-throughput determination of lipid turnover in vivo have been developed based on partial metabolic 2H2O labeling. The performance on lipid kinetics measurement of the proposed methods was validated in three different liquid chromatography-mass spectrometry (LC-MS) setups: MS-only, untargeted MS/MS, and targeted MS/MS. The MS-only scheme consisted of multiple LC-MS runs for quantification of lipid mass isotopomers and an extra LC-MS/MS run for lipid identification. The untargeted MS/MS format utilized multiple data-dependent LC-MS/MS runs for both quantification of lipid mass isotopomers and lipid identification. An in-house software was also developed to streamline the data processing from peak area quantification of mass isotopomers to exponential curve fitting for extracting the turnover rate constant. With HeLa cells cultured in 5% 2H2O media for 48 hr, I could deduce the species-level turnover rates of 108 and 94 lipids in the MS-only and untargeted MS/MS schemes, respectively, which covers 13 different subclasses and spans 3 orders of magnitude. Furthermore, the targeted MS/MS setup, which performs scheduled LC-MS/MS experiments for some targeted lipids, enabled differential measurement between the turnover rates of the head and tail groups of lipid. The reproducibility of the lipid kinetics measurement was also demonstrated with lipids that commonly detected in both positive and negative ion modes or in two different adduct forms.

Chapter 2: Effect of Mass Resolution on Lipid Turnover Rate Measurements A systematic comparison between three types of high-resolution mass spectrometers was performed to determine the effect of mass resolution on lipid turnover rate measurements using partial metabolic heavy water (2H2O) labeling. Lipid Annotator and LipidBlast+MSPePSearch were also compared to determine the optimal lipid identification platform. Lipid Annotator was advantageous for determining retention times of the detected lipids, identifying co-eluted lipids, and obtaining more matched lipids than LipidBlast+MSPePSearch. Using lipid extracts of HeLa cells cultured in 5% 2H2O enriched media for 48 hr and Lipid Annotator software, the turnover rates of individual lipids were measured by three liquid chromatography-mass spectrometers (LC-MS): LC coupled with an Agilent 6520 or 6546 quadrupole-time of flight (Q-TOF) and ultra-high performance liquid chromatography (UPLC) connected with a 15T Fourier transform ion cyclotron (15T FT-ICR) mass spectrometer. The two LC-Q-TOF systems provided ≥ 183 individual lipid turnover rates, of which 90 commonly measured lipids showed good consistency between the two instruments. By distinguishing isobars, isotopic overlap, and isotopic interference in 2H2O labeled lipids from the analyte, use of an Agilent 6546 Q-TOF with a higher mass resolution (100,000 at m/z 723) showed better performance than Agilent 6520 Q-TOF (10,000 at m/z 723). Furthermore, ultra-high resolution FT-ICR (300,000 at m/z 723) suffered adverse effects on turnover rate measurement. Reduction of high mass isotopomer peak intensities in labeled lipids and difficulty of tolerance optimization for chromatogram extraction resulted in detrimental effects on lipid kinetic studies.