Headshot of me in my lab attire in the lab
Author: Alex Van Horn | Major: Chemistry | Semester: Spring 2024
During the spring of 2024, I had the privilege of conducting research within the Chemistry and Biochemistry department under the guidance of Dr. Shang Jia. This project focuses on designing a histidine-specific probe that can catalog and characterize proteins at three distinct levels of complexity. The project begins with model FMOC-amino acids to test the chemoselectivity of the probe, progresses to small model proteins, and then testing on a cell lysate. Over the past semester, I successfully synthesized three different probes (Figure 1) and will be testing them at the first level of complexity this summer.
Figure 1: Synthesis overview for pyrocarbonate probes.
The success of the synthesis was confirmed using 1H NMR and 13C NMR spectroscopy (Figure 2). These results provided a solid foundation to proceed with the next stages of the project.
Figure 2: 1H NMR results for DPPC and DPPC-S1
The chemoselectivity of the probes for histidine will be evaluated using high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). This approach allows us to calculate the ratio of tagged histidine to other amino acids, providing insight into the selectivity and efficiency of our probes. One potential challenge we anticipate is the vulnerability of the synthesized three-carbon probes to hydrolysis by an intermediate in the Click reaction. Should this issue arise, we are prepared to synthesize a new set of probes with four or even five carbon alkyne chains. These longer chains would disrupt the formation of the hydrolysis intermediate, thereby preventing the breakdown of the probe-histidine adduct.
In addition to my lab work, I have also engaged in significant activities outside the lab. I presented a poster on this project at the Honors Day Symposium, marking my first experience with poster presentations. This opportunity allowed me to showcase the progress I’ve made and share insights with peers and faculty. The feedback and interactions during the symposium were invaluable, enhancing my understanding of the project and its broader implications. Furthermore, Dr. Jia and I are actively seeking other symposiums and conferences to present our work, aiming to reach a wider audience and gather diverse feedback.
This project has been both challenging and rewarding, offering me hands-on experience in chemical proteomics. Synthesizing the probes and confirming their structures via NMR was a meticulous process that required precision and attention to detail. Each step, from the initial synthesis to the characterization, has provided a deeper understanding of the underlying chemistry and the practical challenges involved in probe design.
Looking ahead, I am eager to continue this project next semester. The upcoming tests on the first stage of complexity will be crucial in validating our hypotheses and refining our approach. Success in these tests will pave the way for subsequent experiments on model proteins and cell lysates, ultimately contributing to a more comprehensive understanding of histidine’s role in protein function.
In conclusion, my work on activity-based protein profiling of nucleophilic amino acid residues with pyrocarbonates represents a significant step towards developing tools for protein characterization. The synthesis of histidine-specific probes and their subsequent testing will provide insights that could have broader applications in proteomics. I am committed to advancing this research, overcoming challenges, and contributing to the scientific community’s understanding of protein functions. Through continued experimentation and presentation of our findings, I hope to make a meaningful impact in the field of chemical proteomics.