BENJAMIN GARCIA, Ph.D.
Presidential Professor of Biochemistry and Biophysics
Department of Biochemistry and Biophysics
9-124 Smilow Center for Translational Research
3400 Civic Center Blvd.
Perelman School of Medicine
University of Pennsylvania
Philadelphia, PA 19104-6059
215-573-9423 (office); 215-573-9422 (lab)
B.S. University of California, Davis (2000) Chemistry
Ph.D. University of Virginia (2005) Chemistry
NIH NRSA Postdoctoral Fellow, Institute for Genomic Biology Postdoctoral Fellow, University of Illinois, Urbana-Champaign (2008)
DESCRIPTION OF RESEARCH INTERESTS:
Quantitative Mass Spectrometry Based Proteomics for Characterizing Modified Proteins and Proteomes
The sequences of the human genome and genomes of many other organisms are now readily available and have revolutionized modern biological research. Nevertheless, the next challenge presently on the horizon (after the post-genome era) is the comprehensive characterization of cellular proteins (i.e. the “proteome”), the ‘active/expressed’ part of the genome. DNA sequence or mRNA levels alone cannot predict the dynamic aspects of cellular function. Proteins, their post-translational modifications (PTMs) and the multi-protein complexes they form are the driving forces of cellular machinery that control a diverse number of physiological events. These observations have led to the emergence of a new sub-field of contemporary biology called Proteomics: the characterization of the protein complement expressed by a genome of a particular organism, tissue or cell. At the heart of proteomic experiments is the use of nanoflow liquid chromatography-tandem mass spectrometry for the analysis of complex protein mixtures, which is arguably the most rapid, sensitive and accurate technique available for sequence characterization of proteins.
The Garcia laboratory is focused on developing novel mass spectrometry based proteomic methodologies for quantitatively characterizing changes in protein and proteome expression and post-translational modification state during significant biological events, or in response to external perturbation. Our goal is to utilize large-scale proteomic data to improve our understanding of biological processes at the molecular level. Application of our proteomic technology spans several areas of cellular biology, but a couple of main interests are described below.
Towards deciphering the epigenetic Histone Code
Epigenetics refers to stable heritable changes in gene expression that are not due to changes in DNA sequence, such as DNA methylation, RNA interference and histone PTMs. These epigenetic changes are responsible for generating different cell types originating from the exact same genome. Emerging as one key regulator of cellular memory are histones. Histones are small basic proteins that function to package genomic DNA into repeating nucleosomal units (containing ~146 bp of DNA wrapped around two copies each of histones H3, H4, H2A and H2B) forming the chromatin fiber and hence our chromosomes. In general, the packaging of DNA into chromatin is recognized to be a major mechanism by which the access of genomic DNA is restricted. This physical barrier to the underlying DNA is precisely regulated, at least in part, by the PTMs on histones.
A wide number of studies show that several single covalent histone modifications such as methylation, acetylation, phosphorylation and ubiquitination located in the N-terminal tails correlate with both the regulation of chromatin structure during active gene expression, or heterochromatin formation during gene silencing (i.e. the “Histone Code”). Nevertheless, it is currently unknown what effects, if any, multiple combinations of histone modifications might exert, and translating the combinatorial modification patterns of histones into biological significance remains a significant challenge. Additionally, these histone PTMs occur on multiple but specific sites, suggesting that histones can act as signaling platforms for proteins that bind or “read” these marks. In support, several proteins that contain special domains that bind various PTM sites on histones have been discovered.
The Garcia lab has developed proteomics techniques that are considered state of the art for histone PTM analyses and are used world-wide by many research groups. Therefore, we feel that the utilization of advanced proteomic technology in the chromatin biology field will enhance investigations of histone modifications to a much higher scale. In combination with cell and biochemical experimentation, bioinformatics analysis and other “omics” technologies; we feel that our large-scale proteomic data will help provide a systems biology outlook on epigenetic processes that will lay the foundation for development of drug treatments for human diseases that are believed to involve epigenetic mechanisms.
Dynamics of proteome-wide PTM mediated signaling pathways
Another goal of the Garcia lab is to develop and apply novel proteomics based methodology to understand how signaling pathways affect cellular functions and ultimately cell phenotypes. We use quantitative mass spectrometry to measure dynamic changes in protein abundances, protein PTM states, and to characterize protein:protein interactions. For example, we are specifically developing large-scale approaches to isolate and characterize a variety of different types of PTM modified proteomes (e.g. protein phosphorylation, methylation, acetylation, glycosylation, ADP-ribosylation, etc.).
These types of approaches allow for example, the detection of thousands of modified proteins from cells or tissues. When combined with quantitative proteomics techniques such as stable isotope labeling of amino acids in cell culture (SILAC), these cutting-edge tools allow us to examine with unprecedented detail, the molecular level events involved in various biological processes such as during stem cell differentiation, viral infection, metabolic disorder or cancer progression.
We are also very interested in the dynamics of protein modification, and have developed in vivo cellular metabolic labeling strategies to specifically label newly modified proteins. This methodology allows us to determine protein modification turnover rates or kinetics in response to external stimuli. These experiments have allowed us to define for the first time the dynamics of particular classes of modified proteins on a proteome scale. Lastly, we are extremely interested in how these different protein PTM signaling pathways crosstalk with one another, and we are developing the platforms to determine which modifications are found simultaneously on the same proteins, and how this biological code is then transformed to direct a myriad of cellular functions.
- Han Y, Yuan ZF, Molden RC, Garcia BA. (2015) Monitoring cellular phosphorylationsignaling pathways into chromatin and down to the gene level. Mol Cell Proteomics Nov 5. pii: mcp.M115.053421. [Epub ahead of print]
- Sidoli S, Simithy J, Karch KR, Kulej K, Garcia BA.(2015) Low resolution data-independent acquisition in an LTQ-Orbitrap allows for simplified and fully untargeted analysis of histone modifications. Anal Chem. Nov 5. [Epub ahead of print]
- Zhao Y, Garcia BA. (2015) Comprehensive catalog of currently documented histone modifications. Cold Spring Harb Perspect Biol 7(9).
- Sidoli S, Garcia BA. (2015) Properly reading the histone code by MS-based proteomics. Proteomics 15(17):2901-2902.
- Kulej K, Avgousti DC, Weitzman MD, Garcia BA. (2015) Characterization of histone post-translational modifications during virus infection using mass spectrometry-based proteomics. Methods. pii: S1046-2023(15)00249-2.
- Molden RC, Bhanu NV, LeRoy G, Arnaudo AM, Garcia BA. (2015) Multi-faceted quantitative proteomics analysis of histone H2B isoforms and their modifications. Epigenetics Chromatin 8:15.
- Won KJ, Choi I, LeRoy G, Zee BM, Sidoli S, Gonzales-Cope M, Garcia BA. (2015) Proteogenomics analysis reveals specific genomic orientations of distal regulatory regions composed by non-canonical histone variants. Epigenetics Chromatin 8:13.
- Sidoli S, Lin S, Karch KR, Garcia BA. (2015) Bottom-up and middle-down proteomics have comparable accuracies in defining histone post-translational modification relative abundance and stoichiometry. Anal Chem. 87(6):3129-3133.
- Yuan ZF, Lin S, Molden RC, Cao XJ, Bhanu NV, Wang X, Sidoli S, Liu S, Garcia BA. (2015) EpiProfile quantifies histone peptides with modifications by extracting retention time and intensity in high-resolution mass spectra. Mol Cell Proteomics 14(6):1696-1707.
- Bryant JM, Donahue G, Wang X, Meyer-Ficca M, Luense LJ, Weller AH, Bartolomei MS, Blobel GA, Meyer RG, Garcia BA, Berger SL. (2015) Characterization of BRD4 during mammalian postmeiotic sperm development. Mol Cell Biol. 35(8):1433-1448.
- Sidoli S, Lin S, Xiong L, Bhanu NV, Karch KR, Johansen E, Hunter C, Mollah S, Garcia BA. (2015) Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) analysis for characterization and quantification of histone post-translational modifications. Mol Cell Proteomics 14(9):2420-2428.
- Beck DB, Narendra V, Drury WJ 3rd, Casey R, Jansen PW, Yuan ZF, Garcia BA, Vermeulen M, Bonasio R. (2014) In vivo proximity labeling for the detection of protein-protein and protein-RNA interactions. J Proteome Res. 13(12):6135-6143.
- Karch KR, Zee BM, Garcia BA. (2014) High resolution is not a strict requirement for characterization and quantification of histone post-translational modifications. J Proteome Res. 13(12):6152-6159.
- Mews P, Zee BM, Liu S, Donahue G, Garcia BA, Berger SL.(2014) Histone methylation has dynamics distinct from those of histone acetylation in cell cycle reentry from quiescence. Mol Cell Biol.34(21):3968-3980.
- Britton LM, Sova P, Belisle S, Liu S, Chan EY, Katze MG, Garcia BA. (2014) A proteomic glimpse into the initial global epigenetic changes during HIV infection. Proteomics. 14(19):2226-2230.
- Molden RC, Garcia BA.(2014) Middle-down and top-down mass spectrometric analysis of co-occurring histone modifications. Curr Protoc Protein Sci. 77:23.7.1-23.7.28.
- Lin S, Wein S, Gonzales-Cope M, Otte GL, Yuan ZF, Afjehi-Sadat L, Maile T, Berger SL, Rush J, Lill JR, Arnott D, Garcia BA. (2014) Stable-isotope-labeled histone peptide library for histone post-translational modification and variant quantification by mass spectrometry. Mol Cell Proteomics 13(9):2450-2466.
- O'Connor CM, DiMaggio PA Jr, Shenk T, Garcia BA.(2014) Quantitative proteomic discovery of dynamic epigenome changes that control human cytomegalovirus (HCMV) infection. Mol Cell Proteomics. 13(9):2399-2410.
- Molden RC, Goya J, Khan Z, Garcia BA.(2014) Stable isotope labeling of phosphoproteins for large-scale phosphorylation rate determination. Mol Cell Proteomics 13(4):1106-1118.
- Karch KR, Denizio JE, Black BE, Garcia BA. (2013) Identification and interrogation of combinatorial histone modifications. Front Genet. 4:264.
- Britton LM, Newhart A, Bhanu NV, Sridharan R, Gonzales-Cope M, Plath K, Janicki SM, Garcia BA. (2013) Initial characterization of histone H3 serine 10 O-acetylation. Epigenetics 8(10):1101-1113.
- Evertts AG, Manning AL, Wang X, Dyson NJ, Garcia BA, Coller HA. (2013) H4K20 methylation regulates quiescence and chromatin compaction. Mol Biol Cell. 4(19):3025-3037.
- Leroy G, Dimaggio PA, Chan EY, Zee BM, Blanco MA, Bryant B, Flaniken IZ, Liu S, Kang Y, Trojer P, Garcia BA.(2013) A quantitative atlas of histone modification signatures from human cancer cells. Epigenetics Chromatin 6(1):20.
- Sridharan R, Gonzales-Cope M, Chronis C, Bonora G, McKee R, Huang C, Patel S, Lopez D, Mishra N, Pellegrini M, Carey M, Garcia BA, Plath K.(2013) Proteomic and genomic approaches reveal critical functions of H3K9 methylation and heterochromatin protein-1γ in reprogramming to pluripotency. Nat Cell Biol. 15(7):872-882.
- Cao XJ, Arnaudo AM, Garcia BA (2013 Large-scale global identification of protein lysine methylation in vivo. Epigenetic 8(5):477-485.
- Evertts AG, Zee BM, Dimaggio PA, Gonzales-Cope M, Coller HA, Garcia BA.(2013) Quantitative dynamics of the link between cellular metabolism and histone acetylation. J Biol Chem. 288(17):12142-12151.