Molecular Biology Faculty

Benjamin A. Garcia

bagarcia	Garcia Research Lab
(609) 258-8854	Schultz Lab, 415
	Lab (609) 258-3001
Faculty Assistant	Matt Montondo
This email address is being protected from spambots. You need JavaScript enabled to view it.	(609) 258-2664

Research Focus

Quantitative Mass Spectrometry Based Proteomics for Analysis of Eukaryotic Chromatin Structure and Function

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 proteins, 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 the cellular machinery. 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 or tissue. 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 expression and post-translational modification state within a given proteome 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. We currently employ Bottom up (sequencing of small peptides), Middle down (sequencing of large polypeptides) and Top down (sequencing of intact proteins) proteomic strategies and allied techniques to achieve our aims. Application of our proteomic technology spans several areas of molecular biology, but one main interest is described below.

A proteomic approach for systems-wide analysis of key molecular events during epigenetic processes

Epigenetic 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 modifications. These epigenetic changes are responsible for generating different cell types originating from the exact same genome. Additionally, even though all genes exist in every cell, only a small number of genes are expressed in any given cell type and these expression patterns can be "memorized". Inheritance of these transcription patterns through DNA replication and chromatin remodeling that accompanies each cell division is crucial for cell survival, but the mechanisms by which this "memory" is achieved is not well understood. 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 of 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. These histone PTMs occur on multiple but specific sites, suggesting that histones can act as signaling platforms for proteins that "read" these marks. In support, proteins that contain special domains that bind to single site-specific methylation and acetylation marks on histones have been discovered. Consequently, the "Histone Code" hypothesis and other theories have been put forward to explain how these histone marks can result in distinct cellular outcomes in terms of chromatin-regulated functions. 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. Therefore, we feel that the utilization of advanced proteomic technology in chromatin biology will enhance investigations of histone modifications to a much higher scale.

For investigating such a complex mechanism, we are focusing on uncovering changes in "Histone Codes" as a result of exogenous stimuli, over-expression, or knockdown of proteins that affect chromatin remodeling networks. Additionally, we anticipate tracking the chromatin remodeling signaling cue to changes in overall global protein expression, alterations in signal transduction cascades (phosphoproteomics), and disruption of protein-protein interaction networks which will produce information concerning the dynamic mechanism of response in regard to the external perturbation. In combination with 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 and conditions that are believed to be of epigenetic origin.

Selected Publications

Korpal M, Ell BJ, Buffa FM, Ibrahim T, Blanco MA, Celià-Terrassa T, Mercatali L, Khan Z, Goodarzi H, Hua Y, Wei Y, Hu G, Garcia BA, Ragoussis J, Amadori D, Harris AL, Kang Y. (2011) Direct targeting of Sec23a by miR-200s influences cancer cell secretome and promotes metastatic colonization. Nat Med. Aug 7. [Epub ahead of print]

Arnaudo AM, Molden RC, Garcia BA. (2011) Revealing histone variant induced changes via quantitative proteomics. Crit Rev Biochem Mol Biol. 46: 284-294. PubMed

Zee BM, Levin RS, Dimaggio PA, Garcia BA. (2010) Global turnover of histone post-translational modifications and variants in human cells. Epigenetics Chromatin. 3: 22. PubMed

Young NL, Dimaggio PA, Garcia BA. (2010) The significance, development and progress of high-throughput combinatorial histone code analysis. Cell Mol Life Sci. 67: 3983-4000. PubMed

Evertts AG, Zee BM, Garcia BA. (2010) Modern approaches for investigating epigenetic signaling pathways. J Appl Physiol. 109: 927-933. PubMed

Olszewski KL, Mather MW, Morrisey JM, Garcia BA, Vaidya AB, Rabinowitz JD, Llinás M. (2010) Branched tricarboxylic acid metabolism in Plasmodium falciparum. Nature. 466: 774-778. PubMed

Garcia BA. (2010) Mass spectrometric analysis of histone variants and post-translational modifications. Front Biosci (Schol Ed). 1: 142-153. PubMed

Young NL, Plazas-Mayorca MD, Dimaggio PA, Flaniken IZ, Beltran AJ, Mishra N, Leroy G, Floudas CA, Garcia BA. (2010) Collective mass spectrometry approaches reveal broad and combinatorial modification of high mobility group protein A1a. J Am Soc Mass Spectrom. 21: 960-970. PubMed

Young NL, Plazas-Mayorca MD, Garcia BA. (2010) Systems-wide proteomic characterization of combinatorial post-translational modification patterns. Expert Rev Proteomics. 7: 79-92. PubMed

Garcia BA. (2009) What does the future hold for top down mass spectrometry? J Am Soc Mass Spectrom. 21: 193-202. PubMed

Zee BM, Levin RS, Xu B, Leroy G, Wingreen NS, Garcia BA. (2009) In vivo residue-specific histone methylation dynamics. J Biol Chem. 285: 3341-3350. PubMed

Zee BM, Garcia BA. (2009) Electron transfer dissociation facilitates sequencing of adenosine diphosphate-ribosylated peptides. Anal Chem. 82: 28-31. PubMed

Plazas-Mayorca MD, Zee BM, Young NL, Fingerman IM, Leroy G, Briggs SD, Garcia BA. (2009) One-pot shotgun quantitative mass spectrometry characterization of histones. J Proteome Res. 8: 5367-5674. PubMed

Khan Z, Bloom JS, Garcia BA, Singh M, Kruglyak L. (2009) Protein quantification across hundreds of experimental conditions. Proc Natl Acad Sci. 106: 15544-15548. PubMed

Dimaggio PA Jr, Young NL, Baliban RC, Garcia BA, Floudas CA. (2009) A mixed-integer linear optimization framework for the identification and quantification of targeted post-translational modifications of highly modified proteins using multiplexed ETD tandem mass spectrometry. Mol Cell Proteomics. 8: 2527-2543. PubMed

Young NL, Dimaggio PA, Plazas-Mayorca MD, Baliban RC, Floudas CA, Garcia BA. (2009) High-throughput characterization of combinatorial histone codes. Mol Cell Proteomics. 8: 2266-2284. PubMed

Leroy G, Weston JT, Zee BM, Young NL, Plazas-Mayorca MD, Garcia BA. (2009) Heterochromatin protein 1 is extensively decorated with histone code-like post-translational modifications. Mol Cell Proteomics. 8: 2432-2442. PubMed

Garcia BA. (2009) Mass spectrometric analysis of histone variants and post-translational modifications. Front Biosci (Schol Ed). 1: 142-153. PubMed

El Gazzar M, Yoza BK, Chen X, Garcia BA, Young NL, McCall CE. (2009) Chromatin-specific remodeling by HMGB1 and linker histone H1 silence proinflammatory genes during endotoxin tolerance. Mol Cell Biol. 29: 1959-1971. PubMed

Garcia BA, Thomas CE, Kelleher NL, Mizzen CA. (2008) Tissue-specific expression and post-translational modification of histone H3 variants. J Proteome Res. 7: 4225-4236. PubMed

Garcia BA, Pesavento JJ, Mizzen CA, Kelleher NL. (2007) Pervasive combinatorial modification of histone H3 in human cells. Nat Methods. 4: 487-489. PubMed

Pesavento JJ, Garcia BA, Steeky JA, Kelleher NL, Mizzen CA. (2007) Mild performic acid oxidation enhances chromatographic and top down mass spectrometric analyses of histones. Mol Cell Proteomics. 6: 1510-1526. PubMed

Garcia BA, Siuti N, Thomas CE, Mizzen CA, Kelleher NL. (2007) Characterization of neurohistone variants and post-translational modifications by electron capture dissociation mass spectrometry. Intl J Mass Spec. 259: 184-196.

Luo W, Peterson A, Garcia BA, Shabanowitz J, Yost HJ, Hunt DF, Virshup DM. (2007) Protein phosphatase 1 regulates assembly and function of the beta-catenin degradation complex. EMBO J 26: 1511-1521. PubMed

Garcia BA, Mollah S, Ueberheide BM, Busby SA, Muratore TL, Shabanowitz J, Hunt DF. (2007) Chemical derivatization of histones for facilitated analysis by mass spectrometry. Nature Protoc. 2: 933-938. PubMed

Morris SA, Rao B, Garcia BA, Hake SB, Shabanowitz J, Hunt DF, Allis CD, Lieb JD, Strahl BD. (2007) Identification of histone H3 K36 acetylation as a highly conserved modification in eukaryotes. J Biol Chem. 282: 7632-7640. PubMed

Garcia BA, Hake SB, Recht J, Morris SA, Strahl BD, Allis CD, Hunt DF. (2007) Organism-specific post-translational modification differences in histones H3 and H4. J Biol Chem. 282: 7641-7655. PubMed

Garcia BA, Shabanowitz J, Hunt DF. (2007) Characterization of histones and their post-translational modifications by mass spectrometry. Curr Opin Chem Biol. 11: 66-73. PubMed

Hake SB, Garcia BA, Duncan EM, Kauer M, Shabanowitz J, Allis CD, Hunt DF. (2006) Expression patterns and post-translational modifications associated with mammalian histone H3 variants. J Biol Chem. 281: 559-568. PubMed

Garcia BA, Joshi S, Thomas CE, Chitta R, Diaz RL, Andrews PC, Loo RA, Shabanowitz J, Kelleher NL, Mizzen CA, Allis CD, Hunt DF. (2006) Comprehensive phosphoprotein analysis of linker histone H1 from Tetrahymena thermophila. Mol Cell Proteomics. 5: 1593-1609. PubMed

Swiatek W, Kang H, Garcia BA, Coombs GS, Shabanowitz J, Hunt DF, Virshup DM. (2006) Negative regulation of LRP6 function by casein kinase I epsilon phosphorylation. J Biol Chem. 281: 12233-12241. PubMed

Recht J, Tsubota T, Tanny JC, Diaz RL, Berger JM, Zhang X, Garcia BA, Shabanowitz J, Burlingame AL, Hunt DF, Kaufman PD, Allis CD. (2006) Histone chaperone Asf1 is required for histone H3 lysine 56 acetylation, a modification associated with S phase in mitosis and meiosis. Proc Natl Acad Sci USA 103: 6988-6993. PubMed

Garcia BA, Platt MD, Born TL, Shabanowitz J, Marcus NA, Hunt DF. (2006) Protein profile of osteoarthritic human articular cartilage using tandem mass spectrometry. Rapid Commun Mass Spectrom. 20: 2999-3006. PubMed

Li Y, Kao G, Garcia BA, Shabanowitz J, Hunt DF, Qin J, Phelan C, Lazar MA. (2006) A novel histone deacetylase pathway regulates mitosis by modulating Aurora B kinase activity. Genes Dev. 20: 2566-2579. PubMed

Klimowski L, Garcia BA, Shabanowitz J, Hunt DF, Virshup DM (2006) Site-specific casein kinase 1epsilon-dependent phosphorylation of Dishevelled modulates beta-catenin signaling. FEBS J. 273: 4594-4602. PubMed

Yang CS, Vitto MJ, Busby SA, Garcia BA, Kesler CT, Gioeli D, Shabanowitz J, Hunt DF, Rundell K, Brautigan DL, Paschal BM. (2005) Simian virus 40 small t antigen mediates conformation-dependent transfer of protein phosphatase 2A onto the androgen receptor. Mol Cell Biol 25: 1298-1308. PubMed

Garcia BA, Shabanowitz J, Hunt DF. (2005) Analysis of protein phosphorylation by mass spectrometry. Methods. 35: 256-264. PubMed

Hake SB, Garcia BA, Kauer M, Baker SP, Shabanowitz J, Hunt DF, Allis CD. (2005) Serine 31 phosphorylation of histone variant H3.3 is specific to regions bordering centromeres in metaphase chromosomes. Proc Natl Acad Sci USA. 102: 6344-6349. PubMed

Garcia BA, Barber CM, Hake SB, Ptak C,Turner FB, Busby SA, Shabanowitz J, Moran RG, Allis CD, Hunt DF. (2005) Modifications of human histone H3 variants during mitosis. Biochemistry 44: 13202-13213. PubMed

Garcia BA, Smalley DM, Shabanowitz J, Ley K, Hunt DF. (2005) The platelet microparticle proteome. J Proteome Res. 5: 1516-1521. PubMed

Fischle W, Tseng B, Dormann H, Ueberheide BM, Garcia BA, Shabanowitz J, Hunt DF, Funabiki H, Allis CD. (2005) Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation. Nature 438: 1116-1122. PubMed

Garcia BA, Busby SA, Shabanowitz J, Hunt DF, Mishra N. (2005) Resetting the epigenetic histone code in the MRL-lpr/lpr mouse model of lupus by histone deacetylase inhibition. J Proteome Res. 6: 2032-2042. PubMed

Syka JEP, Marto JA, Bai DL, Horning S, Senko SW, Schwartz JC, Ueberheide B, Garcia BA, Busby SA, Muratore T, Shabanowitz J, Hunt DF. (2004) Novel linear quadrupole ion trap/FT mass spectrometer: performance characterization and use in the comparative analysis of histone H3 post-translational modifications. J Proteome Res. 3: 621-626. PubMed

Garcia BA, Busby SA, Barber CM, Shabanowitz J, Allis CD, Hunt DF. (2004) Characterization of phosphorylation sites on human histone H1 isoforms by tandem mass spectrometry. J Proteome Res. 3: 1219-1227. PubMed

Johnson L, Mollah S, Garcia BA, Muratore ML, Shabanowitz J, Hunt DF, Jacobsen SE. (2004) Mass spectrometry analysis of arabidopsis histone H3 reveals distinct combinations of post-translational modifications. Nucleic Acids Res. 32: 6511-6518. PubMed

Tang K, Shahgholi M, Garcia BA, Heaney PJ, Cantor CR, Scott LG, Williamson JR. (2002) Improvement in apparent resolution of oligonucleotides in mass spectrometers using 12C/14N-enriched samples. Anal Chem. 74: 226-231. PubMed

Garcia BA, Heaney PJ, Tang K. (2002) Improvement in the MALDI-TOF analysis of DNA with thin-layer matrix preparation. Anal Chem. 74: 2083-2091. PubMed

Shahgholi M, Garcia BA, Chiu NL, Heaney PJ, Tang K. (2001) Sugar additives for MALDI matrices improve signal allowing the smallest nucleotide change (A:T) in a DNA sequence to be resolved. Nucleic Acids Res. 29: e91. PubMed

Garcia BA, Ramirez J, Wong S, Lebrilla CB. (2001) Thermal dissociation of protonated cyclodextrin-amino acid complexes in the gas phase. Int J Mass Spec. 210: 215-222.

Liu J, Tseng K, Garcia BA, Lebrilla CB, Mukerjee Collins S, Smith R. (2001) Electrophoresis separation in open microchannels. A method for coupling electrophoresis with MALDI-MS. Anal Chem. 73: 2147-2151. PubMed

He F, Ramirez J, Garcia BA, Lebrilla CB. (1999). Differentially heated capillary for thermal dissociation of non-covalently bound complexes produced by electrospray ionization. Int J Mass Spec. 182: 261-273.