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Comparative Proteomics Analysis of Four Commonly Used Methods for Identification of Novel Plasma Membrane Proteins

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Abstract

Plasma membrane proteins perform a variety of important tasks in the cells. These tasks can be diverse as carrying nutrients across the plasma membrane, receiving chemical signals from outside the cell, translating them into intracellular action, and anchoring the cell in a particular location. When these crucial roles of plasma membrane proteins are considered, the need for their characterization becomes inevitable. Certain characteristics of plasma membrane proteins such as hydrophobicity, low solubility, and low abundance limit their detection by proteomic analyses. Here, we presented a comparative proteomics study in which the most commonly used plasma membrane protein enrichment methods were evaluated. The methods that were utilized include biotinylation, selective CyDye labeling, temperature-dependent phase partition, and density-gradient ultracentrifugation. Western blot analysis was performed to assess the level of plasma membrane protein enrichment using plasma membrane and cytoplasmic protein markers. Quantitative evaluation of the level of enrichment was performed by two-dimensional electrophoresis (2-DE) and benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis (16-BAC/SDS-PAGE) from which the protein spots were cut and identified. Results from this study demonstrated that density-gradient ultracentrifugation method was superior when coupled with 16-BAC/SDS-PAGE. This work presents a valuable contribution and provides a future direction to the membrane sub-proteome research by evaluating commonly used methods for plasma membrane protein enrichment.

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Abbreviations

PM:

Plasma membrane

MPs:

Membrane proteins

PMPs:

Plasma membrane proteins

PMAPs:

Plasma membrane-associated proteins

PBS:

Phosphate-buffered saline

TBS:

Tris-buffered saline

HB:

Homogenization buffer

CHO:

Chinese Hamster Ovary

MS:

Mass spectrometry

IEF:

Isoelectric focusing

2-DE:

Two-dimensional electrophoresis

16-BAC/SDS-PAGE:

Benzyldimethyl-n-hexadecylammonium chloride/sodium dodecyl sulfate polyacrylamide gel electrophoresis

LC–MS/MS:

Liquid chromatography-mass spectrometry

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Acknowledgements

This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) under the Grant Number of 113S868.

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Authors and Affiliations

  1. Biomedical Engineering Department, Technology Faculty, Kocaeli University, 41380, Kocaeli, Turkey

    Kubra Karaosmanoglu Yoneten

  2. Department of Medical Biology, School of Medicine, Kocaeli University, 41380, Kocaeli, Turkey

    Murat Kasap, Gurler Akpinar & Aylin Kanli

  3. Department of Histology and Embryology, School of Medicine, Istinye University, 34010, Istanbul, Turkey

    Erdal Karaoz

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  1. Kubra Karaosmanoglu Yoneten
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  2. Murat Kasap
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  3. Gurler Akpinar
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  5. Erdal Karaoz
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Correspondence to Murat Kasap.

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Yoneten, K.K., Kasap, M., Akpinar, G. et al. Comparative Proteomics Analysis of Four Commonly Used Methods for Identification of Novel Plasma Membrane Proteins. J Membrane Biol 252, 587–608 (2019). https://doi.org/10.1007/s00232-019-00084-3

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