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.
Gel-based mass spectrometric analysis of hippocampal transmembrane proteins using high resolution LTQ Orbitrap Velos Pro
Article first published online: 13 AUG 2014
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Special Issue: Focus on Biomedical Research Trends
Volume 14, Issue 17-18, pages 2084–2088, September 2014
How to Cite
Heo, S., Spoerk, S., Birner-Gruenberger, R. and Lubec, G. (2014), Gel-based mass spectrometric analysis of hippocampal transmembrane proteins using high resolution LTQ Orbitrap Velos Pro. Proteomics, 14: 2084–2088. doi: 10.1002/pmic.201400077
Colour Online: See the article online to view Figs. 1−3 in colour.
- Issue published online: 8 SEP 2014
- Article first published online: 13 AUG 2014
- Accepted manuscript online: 10 JUL 2014 11:33AM EST
- Manuscript Accepted: 4 JUL 2014
- Manuscript Revised: 13 MAY 2014
- Manuscript Received: 4 MAR 2014
- Animal proteomics;
- Fourier Transform mass spectrometry;
- LTQ Orbitrap Velos Pro;
- Membrane proteins;
- Mouse brain;
- Transmembrane domains
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).