Proteomic Analysis of Rice Endosperm Cells in Response to Expression of hGM-CSF Junling Luo, † Tingting Ning, † Yunfang Sun, † Jinghua Zhu, ‡ Yingguo Zhu, † Qishan Lin, ‡ and Daichang Yang* ,† Center of Engineering Research of Plant Biotechnology and Germplasm Utilization, Ministry of Education, Department of Genetics, College of Life Sciences, Wuhan University, 430072, P.R. China, and UAlbany Proteomics Facility, Center for Functional Genomics, University at Albany, 1 Discovery Drive, Rensselaer, New York 12144 Received April 17, 2008 The accumulation of significant levels of transgenic products in plant cells is required not only for crop improvement, but also for molecular pharming. However, knowledge about the fate of transgenic products and endogenous proteins in grain cells is lacking. Here, we utilized a quantitative mass spectrometry-based proteomic approach for comparative analysis of expression profiles of transgenic rice endosperm cells in response to expression of a recombinant pharmaceutical protein, human granulocyte-macrophage colony stimulation factor (hGM-CSF). This study provided the first available evidence concerning the fate of exogenous and endogenous proteins in grain cells. Among 1883 identified proteins with a false positive rate of 5%, 103 displayed significant changes (p-value < 0.05) between the transgenic and the wild-type endosperm cells. Notably, endogenous storage proteins and most carbohydrate metabolism-related proteins were down-regulated, while 26S proteasome-related proteins and chaperones were up-regulated in the transgenic rice endosperm. Furthermore, it was observed that expression of hGM-CSF induced endoplasmic reticulum stress and activated the ubiquitin/ 26S-proteasome pathway, which led to ubiquitination of this foreign gene product in the transgenic rice endosperm. Keywords: Quantitative proteomics • Human granulocyte-macrophage colony stimulation factor • Transgenic rice endosperm • Storage protein • Endoplasmic reticulum stress • Ubiquitination Introduction Transgenic plants, seeds, and cultured plant cells are po- tentially the most economical systems for large-scale produc- tion of recombinant proteins for industrial and pharmaceutical uses. Along with the development of biopharming using cereal grain as a bioreactor, technical concerns regarding the fate of pharmaceutical recombinant proteins and endogenous pro- teins have generated research interest. In addition to regulatory concerns and the need to develop more efficient methods for downstream processing of recombinant proteins, there are at least other two important issues: (1) Can we engineer plant cells to manufacture amounts that allow cost-effective produc- tion? (2) Can we engineer plant cells to post-translationally modify recombinant proteins so that they are structurally and functionally similar to the native proteins? At present, only a few recombinant proteins expressed in plant cell are well- investigated, including phytase, 1,2 human serum albumin, 2 human lysozyme, 3 the B subunit of Escherichia coli heat labile enterotoxin, 4 human cytomegalovirus glycoprotein B, 5,6 and recombinant antibodies. 7-10 These studies provide useful information about the cellular localization and trafficking mechanism of recombinant proteins, but the fate of endog- enous proteins has rarely been investigated. Batista et al. first reported a global fate of the transcriptome of transgenic rice expressing a recombinant antibody for cancer treatments. 11 Recently, Van Droogenbroeck et al. reported that overproduc- tion of recombinant scFv-Fc disturbs normal endoplasmic reticulum (ER) retention and protein-sorting mechanisms in the secretory pathway, with aberrant localization of the ER chaperone calreticulin and binding protein (BiP) and the endogenous seed storage protein cruciferin in the periplasmic space. 12 However, information regarding the effects on the proteome of transgenic cells is lacking. The development of systems biology offers new opportuni- ties for appreciating the processes of gene transcription, mRNA stability, processing, translation initiation and efficiency, and protein sorting and trafficking. Whereas microarray technolo- gies provide information about global gene expression with cells, complementary proteomics strategies monitor expression of proteins and their post-translational modifications. Pro- teomics has been successfully applied to areas as diverse as determining the protein composition of organelles, systematic elucidation of protein-protein interactions and the large-scale mapping of protein phosphorylation in response to stimulus. Relative quantification proteomics using higher sensitivity mass spectrometry techniques is becoming increasingly popular due * To whom correspondence should be addressed. Tel: 86-27-6875-4680. Fax: 86-27-6875-4680. E-mail: dyang@whu.edu.cn. † Wuhan University. ‡ University at Albany. 10.1021/pr8002968 CCC: $40.75  XXXX American Chemical Society Journal of Proteome Research XXXX, xxx, 000 A Published on Web 09/09/2008