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Investigating a novel protein using mass spectrometry: the example of tumor differentiation factor (TDF)

Investigating a novel protein using mass spectrometry: the example of tumor differentiation factor (TDF)
Investigating a novel protein using mass spectrometry: the example of tumor differentiation factor (TDF)
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.
0065-2598
509-523
Woods, Alisa G.
edf4f7b3-ab1d-4443-8eaa-6ab2f24114fc
Sokolowska, Izabela
84bfb4df-4577-4497-a906-55043725e3ff
Deinhardt, Katrin
5f4fe23b-2317-499f-ba6d-e639a4885dc1
Darie, Costel C.
d5a4c384-25cc-4211-b991-b4fa8ec5c88b
Woods, Alisa G.
edf4f7b3-ab1d-4443-8eaa-6ab2f24114fc
Sokolowska, Izabela
84bfb4df-4577-4497-a906-55043725e3ff
Deinhardt, Katrin
5f4fe23b-2317-499f-ba6d-e639a4885dc1
Darie, Costel C.
d5a4c384-25cc-4211-b991-b4fa8ec5c88b

Woods, Alisa G., Sokolowska, Izabela, Deinhardt, Katrin and Darie, Costel C. (2014) Investigating a novel protein using mass spectrometry: the example of tumor differentiation factor (TDF). Advances in Experimental Medicine and Biology, 806, 509-523. (doi:10.1007/978-3-319-06068-2_25). (PMID:24952200)

Record type: Article

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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Published date: 2014
Organisations: Biomedicine

Identifiers

Local EPrints ID: 385136
URI: http://eprints.soton.ac.uk/id/eprint/385136
ISSN: 0065-2598
PURE UUID: 28032930-6802-4c35-81f0-78e4e89d2049
ORCID for Katrin Deinhardt: ORCID iD orcid.org/0000-0002-6473-5298

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Date deposited: 15 Jan 2016 15:27
Last modified: 15 Mar 2024 03:45

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Contributors

Author: Alisa G. Woods
Author: Izabela Sokolowska
Author: Costel C. Darie

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