Research Interests

CURRENT RESEARCH

My long-term research interests are to develop and utilize combined approaches in chemistry, biology, and engineering to uncover and understand epigenetic biological mechanisms. The physiologically relevant form of the human genome is determined by chemical modifications on histone packaging proteins and DNA. My research pursues a fundamental understanding of the origins of these modifications and their individual and combinatorial role in driving chromatin structure and gene expression. Toward these efforts, a crucial tool is the ability to synthesize histone proteins with specific chemical modifications, allowing the role of these modifications to be investigated both in vitro and in live cells.

With over a hundred distinct chemical modifications identified on histone proteins, the epigenetic code is dauntingly complex. By combining new developments in chemical biology and protein semi-synthesis with rapidly advancing proteomics and genomics techniques, we are making big strides in our understanding of chromatin biology. Additionally, as a variety of disease states are increasingly found to have epigenetic components, fundamental knowledge of these processes will directly inform efforts to create new classes of therapeutics that target these states.

DOCTORAL RESEARCH

Amidst the current -omics revolution in analytical biology, we are fortunate to have highly robust approaches for making genome- and proteome-wide measurements from biological systems. Despite these capabilities, it remains difficult to pin down the precise timing of various biological happenings inside the cell, details that are often of crucial importance to understanding biological function. This is especially true in the field of proteomics: altering patterns of protein synthesis are a major way that cells adapt and respond to changes in environment, and protein lifetimes can vary greatly. In my doctoral research I developed and applied experimental methods for performing the time-resolved analysis of protein synthesis within cells. The work utilized combined approaches in mass spectrometry-based proteomics, chemical biology, and molecular biology to develop methods for measuring proteome-wide changes in protein synthesis at highly precise time scales. In addition to the development of these techniques, I applied them to understand fundamental biological processes in bacterial communication and gene regulation. Most interesting about this work to me was the capability of new analytical methods to explore previously inaccessible biological questions — developments of this kind are crucial in addressing challenging problems in biology and public health.

Bio-orthogonal non-canonical amino acid tagging (BONCAT) - An experimental tool for time-resolved proteomic analysis

Professor David Tirrell’s group at Caltech has developed a method for the selective analysis of newly synthesized cellular proteins called bio‐orthogonal non‐canonical amino acid tagging (BONCAT). The approach involves the incorporation of synthetic amino acids bearing chemical handles into protein translation, and isolating those proteins for subsequent analysis by mass spectrometry-based proteomics. During my graduate work, I made significant improvements to BONCAT that were essential for the method’s advancement and application in biology. I characterized how non-canonical amino acids perturb the endogenous proteome, a primary concern of our biologist collaborators, and developed strategies to incorporate non-canonical amino acids in a non-perturbative manner (Bagert et al., 2014). I combined BONCAT with a quantitative proteomics technique which allowed both the identification and quantification of newly synthesized proteins. I also demonstrated that BONCAT permits proteomic analysis at very short time scales that are inaccessible to alternative approaches (Bagert et al., 2014). Last, I worked with collaborators to develop cleavable biotin probes for BONCAT purification (Szychowski et al., 2010), and to utilize an engineering protein called an aminoacyl-tRNA synthetase to perform BONCAT in a cell-selective manner in complex mixtures of mammalian cells (Mahdavi et al, 2016).

Bagert, John D, et al. “Time-resolved proteomic analysis of quorum sensing in Vibrio harveyi.”. Chem Sci 73 (2016): , 7, 3, 1797-1806.

Mahdavi, Alborz, et al. “Engineered Aminoacyl-tRNA Synthetase for Cell-Selective Analysis of Mammalian Protein Synthesis.”. J Am Chem Soc 138.13 (2016): , 138, 13, 4278-81.

Szychowski, Janek, et al. “Cleavable biotin probes for labeling of biomolecules via azide-alkyne cycloaddition.”. J Am Chem Soc 132.51 (2010): , 132, 51, 18351-60.

Using BONCAT to undestand the bacterial processes of post-transcriptional gene regulation and quorum sensing

I took the development of BONCAT one step further and explored biological applications in which time resolved proteomic information would be particularly useful. I used BONCAT to study the bacterial post-transcriptional small RNA regulator CyaR, validating previously known targets and identifying several new targets that expanded the functional role of CyaR in carbon metabolism, osmoregulation, and transcriptional regulation (Bagert et al., in revision) I also used BONCAT to characterize the time-dependent transition of the quorum-sensing bacterium Vibrio harveyi from individual- to group-behaviors (Bagert et al., 2016; Feng et al., 2015). This work presents the first time-resolved analysis of the quorum-sensing-regulated proteome. My analysis revealed new proteins with diverse functions that are controlled by quorum sensing, including transcription factors, chemotaxis proteins, transport proteins, type VI secretion, and proteins involved in iron homeostasis. In addition to these biological findings, these works serve as a methodological demonstration of how BONCAT can be used to perform time-resolved proteomic analysis to study dynamic regulatory systems in biology.

Bagert, John D, et al. “A quantitative proteomic method for elucidating the function of bacterial small RNA regulators”. (Submitted).

Bagert, John D, et al. “Time-resolved proteomic analysis of quorum sensing in Vibrio harveyi.”. Chem Sci 73 (2016): , 7, 3, 1797-1806.

Feng, Lihui, et al. “A qrr noncoding RNA deploys four different regulatory mechanisms to optimize quorum-sensing dynamics.”. Cell 160.1-2 (2015): , 160, 1-2, 228-40.