Author ORCID Identifier
0009-0004-2027-4022
Document Type
Dissertation
Date of Award
8-31-2026
Degree Name
Doctor of Philosophy in Chemistry - (Ph.D.)
Department
Chemistry and Environmental Science
First Advisor
Hao Chen
Second Advisor
Edgardo Tabion Farinas
Third Advisor
Yuanwei Zhang
Fourth Advisor
Farnaz A .Shakib
Fifth Advisor
Xianqin Wang
Abstract
A wide variety of biologically important molecules, such as enzymes, antibodies, hormones, transporters and receptors are proteins by composition and they play key roles in biological functions such as cellular regulation, communication, metabolism and physiological function. Protein dysfunctions and abnormalities are associated with numerous diseases, making proteins critical targets for understanding disease mechanisms and developing therapeutic interventions. Protein-based therapeutics such as monoclonal antibodies, hormones and vaccines gained popularity due to their high specificity, efficacy, and ability to treat complex diseases that are often difficult to address with small-molecule drugs.
Given the important role of proteins in a variety of biological processes and their widespread use as therapeutic agents, In-depth protein analysis is essential for understanding biological functions and ensuring the safety, efficacy, and quality of protein-based pharmaceuticals. Mass spectrometry (MS) has emerged as a powerful platform for protein analysis because it not only enables detailed structural characterization but also facilitates accurate and sensitive quantification of proteins. The protein backbone and post-translational modifications can be studied with high degree of accuracy. However, we see two issues in this workflow. Firstly, Standards and isotope-labeled internal standards are needed for the absolute quantitation of the target peptides and proteins. These standards are expensive. On most occasions, the surrogate peptides, especially with post translational modifications (PTMs) are not commercially available and difficult to synthesize. Secondly, in-depth structural characterization of proteins via bottom up and middle down approaches often requires time consuming reduction and enzymatic digestion steps to ensure protein cleavage into subunits or peptides. Chemical artifacts can also be induced due to chemical modifications that can occur in-solution during the long digestion process. Considering these limitations, the development of rapid sample preparation technologies and standard-free protein quantification strategies has become increasingly important. Such advances have the potential to accelerate protein analysis workflows, reduce analytical costs, minimize experimental artifacts, and improve quantitative accuracy.
Accordingly, the overarching goal of this work is to develop innovative methodologies for the rapid characterization and standard-free quantification of peptides and proteins. Specifically, this research seeks to eliminate the reliance on calibration curves and expensive reference standards for quantitative analysis while significantly reducing the sample preparation time required for protein digestion. To achieve these objectives, three research projects are presented in this dissertation, encompassing two emerging analytical technologies. The first project focuses on the development and application of standard-free coulometric mass spectrometry (CMS) for the absolute quantification of peptides and proteins by derivatization. By derivatizing proteins and peptides with electroactive compounds, CMS enables direct quantification without the need for external standards or calibration curves. The second project investigates the ultrafast middle-down digestion of antibody charge variants following separation by strong cation exchange (SCX) chromatography. This approach utilizes the enzymes IdeS and EndoS2 in conjunction with tris(2-carboxyethyl)phosphine (TCEP) to rapidly generate antibody subunits for detailed structural characterization. The third project explores the ultrafast digestion of proteins following size exclusion chromatography (SEC) separation, enabling streamlined protein characterization through the integration of chromatographic separation and rapid enzymatic digestion.
The successful implementation of these technologies demonstrates their potential to improve the speed, efficiency, and cost-effectiveness of protein analysis. Collectively, these advances provide powerful tools for quantitative and structural characterization of proteins and may facilitate broader applications in biological research, clinical analysis, and the development of biopharmaceutical products.
Recommended Citation
Fnu, Praneeth Ivan Joel, "Development of ultrafast protein digestion and standards free quantitation methods for mass spectrometric analysis" (2026). Dissertations. 1896.
https://digitalcommons.njit.edu/dissertations/1896
Included in
Analytical Chemistry Commons, Analytical, Diagnostic and Therapeutic Techniques and Equipment Commons, Biochemistry, Biophysics, and Structural Biology Commons, Chemicals and Drugs Commons
