The New Jersey Institute of Technology's
Electronic Theses & Dissertations Project

Title:	Development of novel protein digestion and quantitation methods for mass spectrometic analysis
Author:	Ai, Yongling
View Online:	njit-etd2023-053 (xxii, 139 pages ~ 5.0 MB pdf)
Department:	Department of Chemistry and Environmental Science
Degree:	Doctor of Philosophy
Program:	Chemistry
Document Type:	Dissertation
Advisory Committee:	Chen, Hao (Committee chair) Sadik, Omowunmi A. (Committee member) Mitra, S. (Committee member) Zhang, Yuanwei (Committee member) Wei, Zhi (Committee member)
Date:	2023-12
Keywords:	Coulometric mass spectrometry Electrochemistry Mass spectrometry Microdroplet reaction Peptide quantitation Protein quantitation
Availability:	Unrestricted
Abstract:	Proteins are the workhorses of biology, playing multifaceted roles in maintaining cellular function, signaling, and response to environmental cues. Understanding their abundance and dynamics is pivotal for unraveling the complexities of biological processes, which underpins the foundations of molecular and cellular biology. Accurate measurement of protein quantities provides insights into cellular homeostasis, facilitates the discovery of biomarkers, and sheds light on the molecular mechanisms of diseases, bridging the gap between the molecular intricacies of proteins and their functional consequences in health and disease. The evolution of protein quantitation methodologies, from classical colorimetric assays to sophisticated mass spectrometry-based approaches, has expanded the analytical precision and dynamic range, enabling the detection of proteins at low abundance levels. The development of targeted proteomic techniques has further refined the quantitation process, allowing researchers to investigate specific proteins or post-translational modifications with high sensitivity and accuracy. However, bottom-up targeted proteomic analysis and quantitation often requires overnight tryptic digestion to ensure complete protein cleavage into peptides, which may significantly hamper experimental efficiency and induce some chemical modifications during the long digestion process. In addition, isotope-labeled internal standards are needed for the absolute quantitation of the target proteins. Syntheses of those standards are time-consuming and costly, and some of standards might not be available or difficult to synthesize if the surrogate peptides contain post-translational modifications (PTMs). Therefore, developing new technologies to speed up the sample preparation process like digestion and standard-free quantitation strategies is essential and holds the promise of unlocking deeper insights into biology, driving innovation in diagnostics, and advancing the development of targeted therapies. Accordingly, the goal of this work is to develop novel methods for the ultrafast tryptic digestion of proteins and standard-free quantitation for peptides and proteins absolute quantitation without building the calibration curve. Three projects are included here with two novel technologies, i.e., ultrafast microdroplet digestion and coulometric mass spectrometry, showing the different applications. Firstly, the investigation of tryptic protein digestion in microdroplets and in bulk solution is conducted to comprehensively evaluate the microdroplet digestion versus bulk overnight digestion by examining digestion efficiency. Secondly, standard-free coulometric mass spectrometry (CMS) is applied for the absolute quantitation of tryptophan-containing peptides and amyloid beta-peptide fragments. Thirdly, coulometric mass spectrometry is further extended for the absolute quantitation of protein mixture sample, host cell proteins as well as deamidation modification. The successful application of those emerging technologies shows the potential for fast and cost-efficient quantitation of peptides and proteins in clinical and pharmaceutical settings.