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

  • Izabela Sokolowska
  • Armand G. Ngounou Wetie
  • Alisa G. Woods
  • Madhuri Jayathirtha
  • Costel C. Darie
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1140)

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 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 immunohistochemistry (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 cells 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.

Keywords

Hormone Growth-factor Neurons CNS Mass spectrometry Proteomics 

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 X glioma cell line

TDF

Tumor differentiation factor

TDF-R

TDF receptor

WB

Western blotting

Notes

Acknowledgements

We would like to thank the past and current lab members for the great working environment.

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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Izabela Sokolowska
    • 1
  • Armand G. Ngounou Wetie
    • 1
  • Alisa G. Woods
    • 1
  • Madhuri Jayathirtha
    • 1
  • Costel C. Darie
    • 1
  1. 1.Biochemistry & Proteomics Group, Department of Chemistry & Biomolecular ScienceClarkson UniversityPotsdamUSA

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