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Investigating a Novel Protein Using Mass Spectrometry: The Example of Tumor Differentiation Factor (TDF)

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Advancements of Mass Spectrometry in Biomedical Research

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 806))

Abstract

Better understanding of central nervous system (CNS) molecules can include the identification of new molecules and their receptor systems. Discovery of novel proteins and elucidation of receptor targets can be accomplished using mass spectrometry (MS). We describe a case study of such a molecule, which our lab has studied using MS in combination with other protein identification techniques, such as immunohistochemistry (IHC) and Western blotting. This molecule is known as tumor differentiation factor (TDF), a recently-found protein secreted by the pituitary into the blood. TDF mRNA has been detected in brain; not heart, placenta, lung, liver, skeletal muscle, or pancreas. Currently TDF has an unclear function, and prior to our studies, its localization was only minimally understood, with no understanding of receptor targets. We investigated the distribution of TDF in the rat brain using IHC and immunofluorescence (IF). TDF protein was detected in pituitary and most other brain regions, in specific neurons but not astrocytes. We found TDF immunoreactivity in cultured neuroblastoma, not astrocytoma. These data suggest that TDF is localized to neurons, not to astrocytes. Our group also conducted studies to identify the TDF receptor (TDF-R). Using LC-MS/MS and Western blotting, we identified the members of the Heat Shock 70-kDa family of proteins (HSP70) as potential TDF-R candidates in both MCF7 and BT-549 human breast cancer cells (HBCC) and PC3, DU145, and LNCaP human prostate cancer cells (HPCC), but not in HeLa cells, NG108 neuroblastoma, or HDF-a and BLK CL.4 cell fibroblasts or fibroblast-like cells. These studies have combined directed protein identification techniques with mass spectrometry to increase our understanding of a novel protein that may have distinct actions as a hormone in the body and as a growth factor in the brain.

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Abbreviations

CNS:

Central nervous system

GFAP:

Glial fibrillary acidic protein

GS9L:

Astrocytoma cell line

IF:

Immunofluorescence

IHC:

Immunohistochemistry

NeuN:

Neuron-specific DNA-binding nuclear protein

NG108-15:

Neuroblastoma × glioma cell line

TDF:

Tumor differentiation factor

TDF-R:

TDF receptor

WB:

Western blotting

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Acknowledgments

We would like to thank Ms. Laura Mulderig, Scott Nichols, and their colleagues (Waters Corporation) for their generous support in setting up the Proteomics Center at Clarkson University. C.C.D. thanks Drs. Thomas A. Neubert (New York University, Belinda Willard (Cleveland Clinic), and Gregory Wolber & David Mclaughin (Eastman Kodak Company) for donation of a TofSpec2E MALDI-MS (each). C.C.D. thanks his advisors, Vlad Artenie, Wolfgang Haehnel, Paul M. Wassarman & Thomas A. Neubert advice and support. This work was supported in part by private donations (Spring Beck Richardson, Guillaume Mimoun, Bob and Karen Brown), the David A. Walsh fellowship, and by the U.S. Army research office (DURIP grant #W911NF-11-1-0304).

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    Authors and Affiliations

    1. Biochemistry & Proteomics Group, Department of Chemistry & Biomolecular Science, Clarkson University, 8 Clarkson Avenue, Potsdam, NY, 13699-5810, USA

      Alisa G. Woods, Izabela Sokolowska & Costel C. Darie

    2. Centre for Biological Sciences, University of Southampton, Life Sciences Building 85, Southampton, SO17 1BJ, UK

      Katrin Deinhardt

    3. Institute for Life Sciences, University of Southampton, Life Sciences Building 85, Southampton, SO17 1BJ, UK

      Katrin Deinhardt

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    1. Alisa G. Woods
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    2. Izabela Sokolowska
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    4. Costel C. Darie
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    Correspondence to Costel C. Darie .

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    Editors and Affiliations

    1. Chemistry and Biomolecular Science Biochemistry and Proteomics Group, Clarkson University, Potsdam, New York, USA

      Alisa G. Woods

    2. Chemistry and Biomolecular Science Biochemistry and Proteomics Group, Clarkson University, Potsdam, New York, USA

      Costel C. Darie

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    Woods, A.G., Sokolowska, I., Deinhardt, K., Darie, C.C. (2014). Investigating a Novel Protein Using Mass Spectrometry: The Example of Tumor Differentiation Factor (TDF). In: Woods, A., Darie, C. (eds) Advancements of Mass Spectrometry in Biomedical Research. Advances in Experimental Medicine and Biology, vol 806. Springer, Cham. https://doi.org/10.1007/978-3-319-06068-2_25

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