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A new method of high-speed cellular protein separation and insight into subcellular compartmentalization of proteins

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Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Transglutaminase (TGM)-2 is a ubiquitous protein with important cellular functions such as regulation of cytoskeleton, cell adhesion, apoptosis, energy metabolism, and stress signaling. We identified several proteins that may interact with TGM-2 through a discovery-based proteomics method via pull down of flag-tagged TGM-2 peptide fragments. The distribution of these potential binding partners of TGM-2 was studied in subcellular fractions separated by density using novel high-speed centricollation technology. Centricollation is a compressed air-driven, low-temperature stepwise ultracentrifugation procedure where low extraction volumes can be processed in a relatively short time in non-denaturing separation conditions with high recovery yield. The fractions were characterized by immunoblots against known organelle markers. The changes in the concentrations of the binding partners were studied in cells expressing short hairpin RNA against TGM-2 (shTG). Desmin, mitochondrial intramembrane cleaving protease (PARL), protein tyrosine kinase (NTRK3), and serine protease (PRSS3) were found to be less concentrated in the 8.5%, 10%, 15%, and 20% sucrose fractions (SFs) from the lysate of shTG cells. The Golgi-associated protein (GOLGA2) was predominantly localized in 15% SF fraction, and in shTG, this shifted to predominantly in the 8.5% SF and showed larger aggregations in the cytosol of cells on immunofluorescent staining compared to control. Based on the relative concentrations of these proteins, we propose how trafficking of such proteins between cellular compartments can occur to regulate cell function. Centricollation is useful for elucidating biological function at the molecular level, especially when combined with traditional cell biology techniques.

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Abbreviations

ARHGAP:

Rho GTPase activating protein

DMSO:

Dimethyl sulfoxide

DES:

Desmin

ERC:

ELKS/RAB6-interacting/CAST family member

GRIK:

Glutamate receptor ionotropic kainite

GOLGA:

Golgi-associated protein

HCE:

Human corneal epithelial

HIVEP:

Human immunodeficiency virus type I enhancer binding protein

ITGB:

Integrin beta

LC:

Liquid chromatography

MS:

Mass spectrometry

NTRK3:

Protein tyrosine kinase

PARL:

Mitochondrial intramembrane cleaving protease

PBS:

Phosphate-buffered saline

PRSS3:

Serine protease

shRNA:

Short hairpin ribonucleic acid

shTG:

TGM-2 silencing short hairpin RNA

SF:

Sucrose fraction

STXBP2:

Syntaxin binding protein

TGM-2:

Transglutaminase-2

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Acknowledgments

Grant support R677/27/2009, NMRC/TCR/002-SERI/2008, NMRC/CSA/013/2009 are gratefully acknowledged.

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Authors and Affiliations

  1. Ocular Wound Healing and Therapeutics Laboratory, Singapore Eye Research Institute, Singapore, 168751, Singapore

    Evelyn Png, WanWen Lan, Melisa Lazaroo, Silin Chen, Lei Zhou & Louis Tong

  2. Singapore National Eye Center, 11 Third Hospital Avenue, Singapore, 168751, Singapore

    Louis Tong

  3. Duke-NUS Graduate Medical School, Singapore, 169857, Singapore

    Louis Tong

  4. School of Anatomy and Human Biology, University of Western Australia, Perth, Western Australia, Australia

    Melisa Lazaroo

Authors
  1. Evelyn Png
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  2. WanWen Lan
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  3. Melisa Lazaroo
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  4. Silin Chen
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  5. Lei Zhou
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  6. Louis Tong
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Corresponding author

Correspondence to Louis Tong.

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Png, E., Lan, W., Lazaroo, M. et al. A new method of high-speed cellular protein separation and insight into subcellular compartmentalization of proteins. Anal Bioanal Chem 400, 767–775 (2011). https://doi.org/10.1007/s00216-011-4810-0

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  • DOI: https://doi.org/10.1007/s00216-011-4810-0

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