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Mass Spectrometry-Based Proteomics Analyses of Integral Membrane Proteins

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Sample Preparation in Biological Mass Spectrometry

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Abstract

The present work compares two proteomics approaches for the analysis of integral membrane proteins (IMPs). Membrane proteins from human U937 monocytes were enriched in IMP following alkaline solvent extraction to solubilize peripheral membrane proteins. Cell extracts of IMPs were separated either by gel electrophoresis followed by in-gel tryptic digestion and liquid chromatography-tandem mass spectrometry (1D-gel-LC-MS/MS) or digested directly with trypsin to release peptides for subsequent analyses by two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). Both approaches yielded reproducible results with 95% of the matched ions showing less than ±1 min variation in retention time and less than 30% variation in intensity for all sample replicates examined. We also observed a similar proportion (~30%) of identified IMPs using both proteomics approaches. However, the gel-free 2D-LC-MS/MS technique led to a higher number of identified proteins (up to 583 for monocyte sample) and an increase in sequence coverage for transmembrane proteins corresponding to 20 μg of total cell extract. The application of label-free quantitative proteomics is also demonstrated for the identification of differentially abundant IMPs from human U937 monocytic cells exposed to phorbol ester. Protein expression profiling experiments enabled the identification of differentially abundant IMPs including cell growth antigen (CD98), the iron trafficking protein (sideroflexin) and LAMP2, a protein which shuttles between the lysosome and the plasma membrane.

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Authors and Affiliations

  1. Caprion Proteomics, Montréal, QC, Canada

    Eric Bonneil, Sylvain Brunet, Michel Jaquinod, Joseph P.M. Hui, Anik Forest & Pierre Thibault

  2. Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, QC, Canada

    Eric Bonneil & Pierre Thibault

  3. Laboratoire d’Etude de la Dynamique des Protéomes, CEA, Grenoble, France

    Michel Jaquinod

  4. Institute for Marine Biosciences NRC, Halifax, NS, Canada

    Joseph P.M. Hui

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  1. Eric Bonneil
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  2. Sylvain Brunet
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  3. Michel Jaquinod
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  4. Joseph P.M. Hui
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  5. Anik Forest
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  6. Pierre Thibault
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Correspondence to Pierre Thibault .

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Editors and Affiliations

  1. , Department of Genetics and Complex Disea, Harvard School of Public Health, Harvard, Huntington Ave., SPH-1 665, Boston, 02115, Massachusetts, USA

    Alexander R. Ivanov

  2. , Research and Development, Pressure BioSciences, Inc., Norfolk Ave. 14, South Easton, 02375, Massachusetts, USA

    Alexander V. Lazarev

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Bonneil, E., Brunet, S., Jaquinod, M., Hui, J.P., Forest, A., Thibault, P. (2011). Mass Spectrometry-Based Proteomics Analyses of Integral Membrane Proteins. In: Ivanov, A., Lazarev, A. (eds) Sample Preparation in Biological Mass Spectrometry. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-0828-0_32

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