Evelyn Wang (Presenter)
University of Texas at Arlington
Bio: I graduated Summa Cum Laude with a Bachelor of Science in Biochemistry and minor in Biology from the University of Texas at Arlington at 2009. I joined the graduate program at 2012 for my PhD degree in Analytical Chemistry from the University of Texas at Arlington. My research focuses on natural product drug discovery using mass spectrometry and intact protein detection, separation, and quantitation using liquid chromatography – triple quadrupole mass spectrometry. I presented my success in research at 12 conferences including various national and international meetings. Recently, I received the Charles K Baker fellowship award for outstanding character and first place in graduate level presentation from the American Chemical Society Meeting in Miniature. My perspective graduation date with my PhD degree is August 2016.
Authorship: Evelyn H. Wang(1), Frances Carroll (2), Peter C. Combe(3), Kevin A. Schug(1)
(1) Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX (2) Restek Corporation, Bellefonte, PA (3) Shimadzu Biotech, Columbia, MD
| Short Abstract There is an increasing demand for protein quantitation in biological fluid for disease detection, protein therapeutics monitoring, and response control during clinical trials. Current triple quadrupole mass spectrometer (QQQ-MS) protein quantitation methods require protein digestion step that is often incomplete thus introduce error. Our method bypasses protein digestion to directly quantify intact proteins on QQQ-MS with multiple reaction monitoring. A series of model proteins are shown to be sensitively and specifically detected, even in the presence of biological matrices. A chromatographic method was developed to address the complex biological matrix for this system. The success of this project can aid clinical diagnostic and treatment advancements. |
Long Abstract
There is an increase in demand for protein detection and quantitation in biological fluid for disease detection, protein therapeutics monitoring, and response control for drug development during clinical trials. Current methods that use highly sensitive triple quadrupole mass spectrometers (QQQ-MS) rely on protein digestion in a bottom-up format. Digestion steps are often incomplete and can introduce errors to protein quantitation. Therefore, absolute protein quantitation is impossible without including expensive isotopic labeled protein standards. We designed a method that bypasses the protein digestion step to directly quantify intact protein on QQQ-MS. Protein standards including myoglobin, cytochrome c, lactalbumin, lysozyme, and ubiquitin, were used to develop an intact protein quantitative method on a Shimadzu LCMS-8050 QQQ-MS through multiple reaction monitoring (MRM). Inconsistent and irreproducible intact protein fragments were initially a challenge, when collision energy was maximized to convert precursor multiply-charged protein signals into product ions. However, a few reproducible and intense product ions were found using a lower collision energy and high collision gas pressure (argon) environment. MRM transitions were developed and calibration curves of the standard proteins were obtained. Calibration curves were generated with respectable linearity (R^2>0.99). To address the complex matrices in biological fluid, a generic reverse phase chromatography method was developed and separation was performed on widepore Restek Viva C4, C8, C18, Biphenyl, and PFP Propyl (2.1 x 100 mm; 5 μm; 300 Å) columns. Prostate specific antigen (PSA) was also included in the study to prove the feasibility of the method for both of the chromatography and mass spectrometry aspects. Specificity of MRM detections were evaluated for urine and plasma matrices. The method is envisioned to be a model for future development of targeted methods for analysis of important disease indicators such as proteins in biological fluids, especially for clinical diagnostic and treatment advancements.
References & Acknowledgements:
The authors gratefully acknowledge instrumentation support from Shimadzu Scientific Instruments, Inc. and research support from Restek Corporation for this work.
| Description | Y/N | Source |
| Grants | yes | Shimadzu Scientific Instrument and Restek Corporation |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |