Gel-based mass spectrometric analysis of hippocampal transmembrane proteins using high resolution LTQ Orbitrap Velos Pro

Authors

  • Seok Heo,

    1. Department of Pediatrics, Medical University of Vienna, Vienna, Austria
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    • Current address: Seok Heo, The Solomon H Snyder Department of Neuroscience and Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

  • Stefan Spoerk,

    1. Institute of Pathology, Medical University of Graz, Core Facility Mass Spectrometry, Center for Medical Research, BioTechMed Omics Center Graz, Graz, Austria
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  • Ruth Birner-Gruenberger,

    1. Institute of Pathology, Medical University of Graz, Core Facility Mass Spectrometry, Center for Medical Research, BioTechMed Omics Center Graz, Graz, Austria
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  • Gert Lubec

    Corresponding author
    1. Department of Pediatrics, Medical University of Vienna, Vienna, Austria
    • Correspondence: Dr. Gert Lubec, Department of Pediatrics, Medical University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria

      E-mail: gert.lubec@meduniwien.ac.at

      Fax: +43-1-40400-6065

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  • Colour Online: See the article online to view Figs. 1−3 in colour.

Abstract

Membrane proteins (MPs) play diverse important roles for physical interactions, cell communication, molecular transport, and signal transduction. Membrane proteins comprise approximately 25∼35% of the genome in living organisms, but there are difficulties in the analysis at the protein chemical level, in particular due to low abundance and limited solubility. Sequence information on membrane proteins and their complexes would be beneficial to elucidate their function. Proteins were extracted from pooled whole mouse brains, enriched membrane fractions were prepared using either two commercially available kits or 6-aminocaproic acid under denaturing or native conditions followed by gel-based proteomic approaches using blue native (BN-) and SDS-PAGE with subsequent in-gel digestion with several proteases, chymotrypsin, trypsin followed by nano-LC-ESI-MS/MS analysis on LTQ Orbitrap Velos Pro. By combining three different extraction methods and two separation methods, 28.39% of proteins were identified as either “integral” or “anchored/integral” MPs based on UniProtKB database searches. MPs with more than six transmembrane domains (TMDs) were identified more efficiently from BN-PAGE separation although a higher number of proteins was identified from SDS-PAGE separation. Comparative analysis of MPs containing TMDs via gel-based LC-MS/MS using BN-PAGE and SDS-PAGE may be useful to increase the number of identified membrane proteins in brain. All MS data have been deposited in the ProteomeXchange with identifier PXD000311 (http://proteomecentral.proteomexchange.org/dataset/PXD000311).

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