INTACT PROTEIN DETECTION, SEPARATION, AND QUANTITATION USING LIQUID CHROMATOGRAPHY - TRIPLE QUADRUPOLE MASS SPECTROMETRY

Author

Evelyn Hsin-Yi Wang

Graduation Semester and Year

2016

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry

Department

Chemistry and Biochemistry

First Advisor

Kevin A Schug

Abstract

There is an increasing demand for protein detection and quantitation in biological fluids for disease detection, protein therapeutics monitoring, and drug development response control. Current methods use highly sensitive and specific triple quadrupole mass spectrometry (QqQ-MS) to quantify protein digest peptides to predict original intact protein concentrations from the sample. The bottom-up protein quantitation format requires protein digestion which is often incomplete and can introduce errors into protein quantitation methods. Therefore, absolute protein quantitation is impossible without including expensive isotopically labeled protein standards. In our lab, a method that bypasses the protein digestion step to directly quantify intact protein on QqQ-MS was developed. Myoglobin, cytochrome c, lactalbumin, lysozyme, and ubiquitin were used as protein standards to prove the principle. An intact protein quantitation method was developed on a Shimadzu LCMS-8050 QqQ-MS using multiple reaction monitoring (MRM) mode. MRM transitions for all protein standards were developed and calibration curves were obtained with respectable linearity (R2>0.99). To address the complex biological matrices, a generic reversed-phase chromatography method was developed. Retention characteristics on C4, C8, C18, Biphenyl, and PFP Propyl stationary phases were evaluated. Prostate specific antigen (PSA) was also included in the study to prove the feasibility of the method being used for biomarker discovery and quantitation. Specificity of the MRM detection was evaluated for urine and plasma matrices. To more fully investigate gas phase interactions in a triple quadrupole instrument, ion scattering effects, mass resolution filters, and proton transfer were investigated and could be used to optimize future applications. The intact protein quantitation method using QqQ-MS was designed as a model for future development of targeted methods, especially for clinical diagnostic and treatment advancements.

Keywords

Intact proteins, Protein quantitation, Triple quadrupole mass spectrometry, Protein separation, Ion transmission

Disciplines

Chemistry | Physical Sciences and Mathematics

License

This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

Comments

Degree granted by The University of Texas at Arlington

Recommended Citation

Wang, Evelyn Hsin-Yi, "INTACT PROTEIN DETECTION, SEPARATION, AND QUANTITATION USING LIQUID CHROMATOGRAPHY - TRIPLE QUADRUPOLE MASS SPECTROMETRY" (2016). Chemistry & Biochemistry Dissertations. 230.
https://mavmatrix.uta.edu/chemistry_dissertations/230