UCR

Institute for Integrative Genome Biology



Proteomics Core


Research Highlights

The following projects are examples of research employing proteomic approaches.

Natasha Raikhel, Department of Botany & Plant Sciences

The MS proteomics approach was employed in Natasha Raikhel's lab to study the Arabidopsis vegetative vacuolar proteome. Several hundred proteins including both soluble and membrane proteins were identified to reside in this organelle including many functionally unknown proteins. This study revealed an important finding that a SNARE-pin complex function in vacuole. Novel protein candidates involved in protein trafficking pathways such as CTPP or NTPP were also discovered, which may serve as markers for study of basic mechanisms of the CTPP and NTPP pathways. In addition, Dr. Raikhel’s laboratory also compared vacuolar proteome between wild-type and a vpe mutant. A label-free quantitative proteomics method has allowed her lab to discover, in addition to many other proteins affected by the mutant, an important apoptosis regulatory protein that may play a role in the VPE-mediated programmed cell death in plants. Her lab also uses proteomics approach to identify protein targets of small molecules in the area of chemical genomics.

Publications:

S. Pan, C. J. Carter, and N. V. Raikhel (2005). Understanding protein trafficking in plant cells   through proteomics. Expert Review Proteomics 2(5):781-792. HubMed

C. Carter, S. Pan, J. Zouhar, E. L. Avila, T. Girke, and N. V. Raikhel (2004). The  vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins. Plant Cell 16(12):3285–3303. HubMed

E. Rojo, R. Martin, C. Carter, J. Zouhar, S. Pan, J. Plotnikova, H. Jin, M. Paneque, J. J. Sanchez-Serrano, B. Baker, F. Ausubel, N. Raikhel (2004). VPEγ exhibits a caspase-like activity that contributes to defense against pathogens. Current Biology 14(21):1897-1906. HubMed

Julia Bailey-Serres, Department of Botany & Plant Sciences

Dr. Bailey-Serres’ laboratory has used the MS technology to characterize the protein components of plant ribosomes. This resulted in the identification of a protein that was not previously known to be associated with plant ribosomes. In addition, potential PTM sites of plant ribosomal proteins were also discovered with the MS methods. Currently, the lab is taking the proteomics approach to characterize Arabidopsis proteins with no known functions.

Publications:

Chang, I.F., Szick-Miranda, K., Pan S. and Bailey-Serres, J. (2005) Proteomic characterization of evolutionarily conserved and variable proteins of Arabidopsis thaliana cytosolic ribosomes. Plant Physiology. 137(3):848-862. HubMed

Zanetti, M.E., Chang I.-.F, Galbraith D.W., Bailey-Serres, J. (2005) Immuno-affinity purification of polyribosomal complexes of Arabidopsis for global analysis of gene expression. Plant Physiology. 138:624-635. HubMed

Ernest Martinez, Department of Biochemistry

Dr. Martinez’s laboratory has extensively used the MS instruments in the W.M. Keck Proteomics Laboratory to study post-translational modifications (PTM) of the human transcription factor Myc/Max heterodimer. For mapping the post transcriptional modification sites of the Myc and Max proteins, the high mass accuracy of the MS instruments in the lab has enabled Dr. Martinez’s lab to discriminate between modified and non-modified amino acid residues so that different types of modification on specific residues can be precisely determined. With that, Myc was found to be hyper-acetylated in vivo at lysines of five distinct sites. Max was also modified in vivo by both acetylation and phosphorylation at multiple residues of lysine and serine. These findings through MS proteomics have made it possible for his lab to carry out site-mutagenesis analysis to address regulatory roles of these modifications. It has been demonstrated that acetylation regulates the protein turn-over of the Myc and nuclear localization of the Max, respectively. Another on-going project is to discover transcription co-factors and complexes that are important in transcriptional regulation in human cells by MS.

Publications:

Faiola F, Wu Y, Pan S, Zhang K, Farina A, Martinez E (2007). Max is acetylated by p300 at several nuclear localization residues. Biochemical J. 403(3):397-407. HubMed

F. Faiola, X. Liu, S. Lo, S. Pan, K. Zhang, E. Lymar, A. Farina, and E. Martinez (2005). Dual regulation of c-Myc by p300 via acetylation-dependent control of Myc protein turnover and coactivation of Myc-induced transcription. Molecular Cellular Biology  25(23):10220-10234. HubMed

Yinsheng Wang, Department of Chemistry

 

The W.M. Keck Proteomics Laboratory, especially the MALDI-MS/MS system, has greatly assisted the protein/peptide projects in Dr. Wang’s laboratory. This work has identified the sites of phosphorylation of a plant dehydrin protein DHN1 by a combination of b-elimination and MALDI-MS/MS.  In this respect, his laboratory found that seven consecutive serine residues in this protein can all be phosphorylated. Dr. Wang’s lab also studied the nature of arginine methylation of human chromosomal high-mobility group A1a (HMGA1a) protein. The MALDI-MS/MS analyses allowed the lab to not only assign the sites of modification, but also determine that both isoforms of dimethylarginines, i.e., symmetric and asymmetric dimethylarginines, are present in the same protein. The laboratory has discovered a number of new sites of post-translational modifications in HMGA1a and HMGA1b proteins isolated from human breast cancer tissues. Moreover, his lab has investigated the sites and nature of cysteine and methionine oxidation of yeast alcohol dehydrogenase I by a combination of MALDI-MS/MS and LC-MS/MS, finding that one of the cysteine residues in this protein was very susceptible to hydrogen peroxide oxidation to give rise to cysteine sulfonic acid. The extent of oxidation was well correlated with the loss of the enzymatic activity of the protein, suggesting a regulatory role of this cysteine oxidation in enzyme function.

Publications:

Zou Y, Webb K, Perna AD, Zhang Q, Clarke S, Wang Y (2007). A mass spectrometric study on the in vitro methylation of HMGA1a and HMGA1b proteins by PRMTs: methylation specificity, the effect of binding to AT-rich duplex DNA, and the effect of C-terminal phosphorylation. Biochemistry, 46, 7896-7906. HubMed

Zou Y, Wang Y (2007).  Mass spectrometric analysis of high-mobility group proteins and their post-translational modifications in normal and cancerous human breast tissues. J Proteome Res. 6, 2304-2314. HubMed

Men L, Wang Y (2007). The oxidation of yeast alcohol dehydrogenase-1 by hydrogen peroxide in vitro. J Proteome Res. 6, 216-225. HubMed

Jiang X, Wang Y (2006). Acetylation and phosphorylation of high-mobility group A1 proteins in PC-3 human tumor cells. Biochemistry, 45, 7194-7201. HubMed

Zou Y, Wang Y (2005). Tandem mass spectrometry for the examination of the posttranslational modifications of high-mobility group A1 proteins: symmetric and asymmetric dimethylation of Arg25 in HMGA1a protein. Biochemistry, 44, 6293-6301. HubMed

Jiang X, Wang Y (2004).dBeta-elimination coupled with tandem mass spectrometry for the identification of in vivo and in vitro phosphorylation sites in maize dehydrin DHN1 protein. Biochemistry, 43, 15567-15576. HubMed

Jian-Kang Zhu, Department of Botany & Plant Sciences

Knowledge of protein-protein interaction in vivo provides scientists with clues of the proteins’ function. Proteomics studies of cellular proteins enable scientists to find the binding partners of proteins in vivo and to estimate the binding strength. Dr. Zhu’s laboratory has used MS proteomics to identify proteins associated with SOS2, which is one of the most important enzymes involved in salt tolerance in Arabidopsis. Several proteins containing vacuolar H+-ATPase (V-ATPase) were identified to be associated with SOS2, in agreement with the results obtained by the yeast two-hybrid approach. However, the MS method was able to further show that the interaction between SOS2 and V-ATPase was enhanced by salt stress. Functional assays indicated that the H+ transport activity in vacuolar membrane isolated from sos2 mutant plants was less than that from wild type plants, suggesting that SOS2 regulates V-ATPase activity in cells. The mass spectrometry data were indispensable for the project because they provided the initial clues for these findings. In follow-up work, the lab will prepare several tagged proteins for the future mass spectrometry analysis. The proteomics equipment has been essential for the advance of these projects.

Publication:

G. Batelli, P. E. Verslues, F. Agius, Q. Qiu, S. Pan, K. Schumaker, S. Grillo and J. Zhu (2007). SOS2 promotes salt tolerance in part by interacting with the vacuolar H+-ATPase and upregulating its transport activity. Mol Cell Biol 27(22): 7781-90. HubMed

SELECTED PUBLICATIONS:

Listed below are additional publications that used the services of the UCR Noel T. Keen Hall Proteomics Core in their research.

Sohn E, Rojas-Pierce M, Pan S, Carter C, Serrano A, Madueño F,  Rojo E, Surpin M, Raikhel NV (2007). The Shoot Meristem Identity Gene TFL1 Demonstrates a Role for the Protein Storage Vacuole in Plant Development. Proc Natl Acad Sci USA 104(47): 18801-6 Hub Med

S. Pan and N. Raikhel (2008). Unraveling plant vacuoles by proteomics (book chapter, in press).

Pan S, Carter C, and Raikhel NV (2005). Understanding protein trafficking in plant cells through proteomics. Expert Review of Proteomics. 2(5):781-792. HubMed

Chang I-F, Szick-Miranda K, Pan S, and Bailey-Serres J (2005). Proteomic characterization of evolutionarily conserved and variable proteins of Arabidopsis thaliana cytosolic ribosomes.  Plant Physiology 137(3):848-862. HubMed

Carter C, Pan S, Zouhar J, Avila EL, Girke T, and Raikhel NV (2004). The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins. Plant Cell 16(12):3285–3303. HubMed

Rojo E, Martin R, Carter C, Zouhar J, Pan S, Plotnikova J, Jin H, Paneque M, Sanchez-Serrano JJ, Baker B, Ausubel F, Raikhel NV (2004). VPEγ exhibits a caspase-like activity that contributes to defense against pathogens. Current Biology 14(21):1897-1906. HubMed


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