Membrane proteins (MPs) are under-represented in global proteomes due to difficulties involved in analysis.1 The more transmembrane domains (TMDs) an MP has, the greater its hydrophobicity, which in turn affects solubility and challenges analysis. Given the overall importance of MPs in cell interactions and communication, molecular transport, and signal transduction, improved analysis techniques would unlock significant information regarding their functions.
Heo et al. (2014) performed a comparative study of three extraction and two separation methods on mouse brains.2 In doing so, they identified a methodology for more efficient identification of MPs. The researchers prepared enriched membrane fractions from pooled whole mouse brains. From there they extracted MPs using a commercial membrane protein extraction kit under native conditions and another under denaturing conditions, and by ultracentrifugation under native conditions. They then separated the resulting proteins from the native condition extractions using BN-PAGE (Blue Native–polyacrylamide gel electrophoresis). The researchers used a second method, SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) to separate the proteins resulting from extraction under denaturing conditions and the other half of the proteins from ultracentrifugation extraction.
Heo et al. subsequently excised and digested the resulting bands for liquid chromatography–tandem mass spectrometry (LC-MS/MS) using a Dionex UltiMate 3000 LC system. They ionized the sample in the nanospray source with stainless steel emitters and performed analysis in an Orbitrap Velos Pro hybrid ion trap-Orbitrap mass spectrometer (Thermo Scientific). Searching the UniProtKB Mus musculus database, the team then analyzed the MS data.
The researchers found that the denaturing extraction kit followed by SDS-PAGE separation identified the highest number of proteins due to the denaturing conditions utilized in the experiment and the superior resolving power of SDS-PAGE. However, BN-PAGE possessed a higher identification rate of TMD-containing proteins. The authors suggest that BN-PAGE is more efficient for hydrophobic membrane protein separation because of the greater identification rate. In total, they were able to extract 1,878 proteins. MPs containing at least one TMD accounted for 516 of the proteins, while the team identified 17 proteins as membrane-anchored or integral membrane proteins with no confirmed or predicted TMDs.
In summary, the investigators more efficiently identified MPs with >6 TMDs using BN-PAGE separation but a higher number of proteins utilizing SDS-PAGE separation. Taken together, the findings from the comparison of techniques for membrane protein extraction and identification with MS characterization suggest a beneficial method for studying MPs.
1. Speers, A.E., & Wu, C.C. (2007) “Proteomics of integral membrane proteins: Theory and application,” Chemical Reviews, 107 (pp. 3687–714).
2. Heo, S., et al. (2014) “Gel-based mass spectrometric analysis of hippocampal transmembrane proteins using high resolution LTQ Orbitrap Velos Pro,” Proteomics, 14(17–18) (pp. 2084–8).
Post Author: Miriam Pollak. Miriam specialised in neuroscience as an undergraduate but traded in lab work for a post graduate degree in science communication.
She has since had a career that has spanned science communication, science education and communications management.
However, Miriam has found her bliss balancing her love of writing and disseminating medical research with managing a multimillion dollar research budget for a childhood cancer charity in Australia.
Oh, and because she’s a sucker for punishment… she’s just started a Masters in Human Nutrition…