The lab uses proteomics to characterize the proteins of the mammalian cell by a strategy known as the CellMap. The cell is deconvoluted as a consequence of its constituent organelles which are isolated by exhaustive purification and presented to the tandem mass spectrometer. The tandem mass spectrometer is used much as a high resolution “protein” microscope to localize proteins quantitatively in the cell. In Gilchrist et al. (1), we were able to use a label free quantitative approach (2, 3) which considers the stochastic aspect of tandem mass spectrometry to localize quantitatively over 2000 different proteins.
Current work focuses on an extension of CellMap proteomics (4, 5) to organelles in different cell types which differ markedly in their morphological features and function in order to gain mechanistic insight into organelle identity, biogenesis and membrane trafficking mechanisms.
A benefit of proteomics is the uncovering of proteins of unknown function. Proteomics has enabled the localization of over 220 of these (324 have been found) and clustering methodologies have assigned each of these to predicted functional categories which are awaiting validation.
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Calnexin is an example of a novel protein uncovered by a screen for phosphorylated substrates of kinases resident for the endoplasmic reticulum (6, 7). The lab continues to follow a trajectory of understanding this protein mechanistically from its original cDNA cloning and naming to its elucidation as a molecular chaperone with specificity for N-linked glycoproteins (8). The direct visualization of its interaction with its glucosyl linked substrate was deduced by X-ray crystallography (9). This established its mechanism as a lectin but with a long arm emanating from the body of the luminal globular domain of calnexin which is speculated to cradle the substrate polypeptide as well as to deliver the protein folding enzyme, erp57 to the substrate polypeptide (10). Current work is focused on the regulation by calnexin of ER retention of misfolded glycoproteins and how this can be targeted for therapeutic consideration for protein misfolding diseases.
The validation of the functions of the hundreds of ‘novel’ proteins uncovered by proteomics and assigned to the endoplasmic reticulum and Golgi apparatus is an ongoing effort. Proteomics is also used in the context of mouse models of liver disease to elucidate proteins of the cell surface and endosomal apparatus linked to diseases such as diabetes and cancer.
1. Gilchrist, A., C.E. Au, J. Hiding, A.W. Bell, J. Fernandez-Rodriguez, S. Lesimple, H. Nagaya, L. Roy, S.J. Gosline, M. Hallet, J. Paiement, R.E. Kearney, T. Nilsson and J.J.M. Bergeron. 2006. Quantitative proteomics analysis of the secretory pathway. Cell. 127:1265-1281.
2. Blondeau, F., B. Ritter, P.D. Allaire, S. Wasiak, M. Girard, N.K. Hussain, A. Angers, V. Legendre-Guillemin, L. Roy, D. Boismenu, R.E. Kearney, A.W. Bell, J.J.M. Bergeron, and P.S. McPherson. 2004. Tandem mass spectrometry analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling. Proc. Natl. Acad. Sci. USA. 101:3833-3838
3. Bergeron, J.J.M. and M. Hallett. 2007. Peptides you can count on. Nature Biotechnology (News and Views). 25:61-62.
4. Yates, J.R III, A. Gilchrist, K. Howell, and J.J.M. Bergeron, 2005. Proteomics of organelles and large cellular structures. Nature Reviews Molecular Cell Biology. 6:702-714.
5. Au, C.E., A.W. Bell, A. Gilchrist, J. Hiding, T. Nilsson, and J.J.M. Bergeron. 2007. Organellar proteomics to create the CellMap. Curr. Opin. Cell Biol. (in press)
6. Rindress, D., X. Lei, J.P.S. Ahluwalia, P. Cameron, A. Fazel, B.I. Posner, and J.J.M. Bergeron. 1993. Organelle specific phosphorylation: Identification of unique membrane phosphoproteins of the endoplasmic reticulum and endosomal apparatus. J. Biol. Chem. 268:5139-5147.
7. Wada, I., D. Rindress, P.H. Cameron, W-J. Ou, J-J. Doherty, II, D. Louvard, A.W. Bell, D. Dignard, D.Y. Thomas, and J.J.M. Bergeron. 1991. SSRalpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J. Biol. Chem. 266:19599-19610.
8. Ou, W-J., P.H. Cameron, D.Y. Thomas, and J.J.M. Bergeron. 1993. Association of folding intermediates of glycoproteins with calnexin during protein maturation. Nature. 364:771-776.
9. Schrag, J.D., J.J.M. Bergeron, Y. Li, S. Borisova, M. Hahn, D.Y. Thomas, and M. Cygler. 2001. The structure of calnexin, an ER chaperone involved in quality control of protein folding. Molecular Cell. 8:633-644.
10. Schrag, J.D., D.O. Procopio, M. Cygler, D.Y. Thomas, and J.J.M. Bergeron. 2003. Lectin control of protein folding and sorting in the secretory pathway. Trends in Biochemical Sciences. 28:49-57.