OverviewPurpose: Isolation of multi-spanning membrane proteins using a sequential detergent extraction method for downstream analysis by western blotting, immunoprecipitation, and mass spectrometry.

Methods: Membrane proteins were extracted from six cell lines and three tissue types using commercially available membrane protein extraction reagents (Thermo Scientific M-PER™ Mammalian Protein Extraction Reagent, Thermo Scientific Mem-PER™ Plus Membrane Protein Extraction Kit, EMD Millipore ProteoExtract ® Transmembrane Protein Extraction Kit TM-PEK, Bio-Rad ReadyPrep™ Protein Extaction Kit Membrane II). These reagents were benchmarked for the solubilization of multi-spanning integral membrane proteins and evaluated by western blot and mass spectrometry. Extraction efficiency of several multi-spanning membrane proteins including ADP/ATP Translocase 3 (SLC25A6), Sodium/potassium-transporting ATPase subunit alpha-1 (AT1A1), and Adenylate cyclase 2 (ADCY2) was measured by densitometry using the Thermo Scientific myECL Imager. Membrane fractions from HEK293 cells were evaluated by mass spectrometry and integral membrane proteins were annotated using the Uniprot database. To determine compatibility of detergent solubilized membrane fraction with immunoprecipitation, the Sodium potassium ATP transport complex was isolated using an antibody against the beta-1 subunit of the complex.

Results: The use of a sequential detergent extraction method resulted in enrichment of integral multi-spanning membrane proteins compared to whole cell lysates and other non-detergent based membrane protein isolation kits. Western blot densitometry showed higher solubilization of integral membrane proteins with a sequential detergent based method. A higher percentage of integral membrane proteins were also identified using this method. Finally, intact Sodium potassium ATP transporter complex was isolated from the membrane fraction using this method, indicating that the sequential detergent extraction method allows for the isolation of intact membrane protein complexes.

IntroductionMembrane proteins are essential in most cellular processes. Examining the membrane proteome is vital to understanding their role in normal and disease function. However, the isolation and extraction of multi-spanning membrane proteins (>2 transmembrane domains) for proteomic study often proves difficult. Traditional methods for isolating membrane proteins are tedious and time-consuming; requiring ultracentrifugation, mechanical disruption, and expensive equipment. Additional drawbacks of these methods include poor solubilization, incompatibility with downstream applications, and disruption of membrane protein complexes. Using a mild detergent-based, selective extraction protocol (Figure 1) eliminates the hassle of membrane protein isolation, and allows for better reproducibility and higher throughput.

Efficient and Convenient Enrichment of Multi-spanning Membrane Proteins for Proteomic Studies

Joanna Geddes¹, Scott Meier¹, Evelina Cirbaite², Juozas Siurkus², Kay Opperman¹, Barbara Kaboord¹ ¹Thermo Fisher Scientific, Rockford, IL, USA; ²Thermo Fisher Scientific, Vilnius, Lithuania

Conclusions• Multi-spanning membrane proteins are more efficiently extracted using Thermo Scientific Mem-PER Plus Membrane Protein Extraction Kit and can be improved with the addition of salt.

• Chloroform/Methanol/Water clean-up after extraction with Mem-PER Plus improved identification and sequence coverage of integral membrane proteins by LC-MS analysis.

• The non-denaturing detergent used to solubilize membrane proteins does not disrupt membrane protein complexes based on the co-immunoprecipitation of Sodium potassium ATPase alpha and beta subunits

References1. Tokhtaeva, E. et al. Subunit Isoform Selectivity in Assembly of Na,K-ATPase α-β Heterodimers. (2012) The Journal of Biological Chemistry  287, 26115-26125.

Methods Membrane Protein Extraction

Membrane proteins were extracted from 5x106 cultured cells or 50mg of tissue according to protocols outlined in commercially available kits. Protein in each fraction was estimated using the Thermo Scientific BCA assay kit, and lysates were stored at -80oC before use.

Western Blot

Normalized samples were separated on a 4-20% Tris Glycine gel and transferred to nitrocellulose membrane via the Pierce G2 Fast Blotter. Membranes were then blocked, incubated in primary antibody (Thermo Scientific ADCY2 Antibody, Thermo Scientific SLC25A6 Antibody, and CST Na, K-ATPase 1 Antibody) for one hour at room temperature or overnight at 4ºC, washed, incubated in secondary antibody for 30 minutes at room temperature, washed, and incubated for 5 minutes in Thermo Scientific SuperSignal™ West Dura Extended Duration Substrate. Blots were then exposed to film or imaged on the Thermo Scientific myECL™ Imager.

Immunoprecipitation

HeLa cell membrane fraction generated with Mem-PER Plus (125g) was immunoprecipitated with Thermo Scientific Anti-AT1B1 antibody (5g) and Thermo Scientific Pierce Protein A/G Magnetic Beads using MS compatible wash and elution buffers. Eluted samples were dried down, and stored at 4oC.

Mass Spectrometry

HEK293 membrane fractions prepared with commercially available reagents were cleaned up using methanol/chloroform/water extraction, then reduced, alkylated and trypsin digested. The samples were analyzed by LC-MS/MS using the Thermo Scientific VELOS Pro Orbitrap mass spectrometer. IP samples were processed with an in-solution digestion method before MS analysis. Briefly, IP eluates were reconstituted in 6M Urea, 50mM Tris-HCl, pH 8, followed by reduction, alkylation and trypsin digestion overnight. The samples were analyzed by LC-MS/MS using a Thermo Scientific Fusion Orbitrap.

Data Analysis

MS data were analyzed with Thermo Scientific Proteome Discoverer 1.4, searching a custom Human/Mouse/Rabbit protein database with the SEQUEST™ algorithm (<2 missed cleavages per peptide, FDR<0.1). Protein accessions from membrane fractions of HEK293 cells were annotated for gene ontology using QuickGO GO:0016021 integral component of membrane.

Results

SEQUEST is a registered trademark of the University of Washington. ProteoExtract is a trademark of EMD Merck/Millipore. ReadyPrep is a trademark of Bio-Rad. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

C= Cytoplasmic Fraction, M= Membrane Fraction, P= Insoluble Fraction

FIGURE 1. Schematic of membrane protein extraction protocol.

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FIGURE 3. Higher extraction efficiency of multi-spanning integral membrane protein using sequential detergent extraction method

Integral membrane proteins Sodium potassium ATPase alpha 1 (AT1A1) and ADP/ATP Translocase 3 (SLC25A6) were enriched using sequential detergent extraction method (Mem-PER Plus) and compared to non-detergent based methods (TM-PEK A and B) and sodium carbonate methods (ReadyPrep II).

FIGURE 2. Mem-PER Plus solubilizes Adenylate Cyclase 2 from mouse kidney with equal or better efficiency than other detergent mixes

Mouse kidney tissue (50g) was homogenized in the cell permeabilization buffer included in the Mem-PER Plus kit, and subsequently solubilized with either the solubilization buffer included or a combination of detergents.

FIGURE 5. LC-ESI MS/MS analysis of membrane fractions from commercially available membrane protein extraction kits

Table. Peptide digests from equal volumes of whole cell (M-PER) and membrane fractions (Mem-PER Plus, TM-PEK A, TM-PEK B, ReadyPrep II) from HEK293 analyzed on a Thermo Scientific Velos Pro Orbitrap. Sequential detergent solubilized membrane fractions (Mem-PER Plus) had a higher total of integral membrane proteins identified based on GOTERM: 0016021. Figure. Sequential detergent extraction increases enrichment for integral and peripheral membrane proteins compared to non-detergent methods and whole cell lysates. Integral membrane proteins ranging from 1-12 transmembrane domains had higher sequence coverage with Mem-PER Plus compared to other commercial reagents

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Table 2: Identification of peptides by LC-MS/MS from immunoprecipitated Sodium potassium ATP transport complex extracted with Mem-PER Plus

Samples were processed by in-solution digestion according to the kit protocol, and analyzed on the Fusion Orbitrap.

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FIGURE 5. Sodium potassium ATP Transport Complex

Sodium potassium ATP Transport complex consists of catalytic subunit alpha and regulatory subunit beta which are both essential for function, as well as an adaptor/regulatory FXYD protein (gamma subunit). This complex pumps sodium out of the cell and potassium into the cell as a function of ATP hydrolysis. (1).

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FIGURE 4. Increased extraction efficiency of multi-spanning integral membrane protein with an isotonic solution

HEK293 cells (5x10⁶) were lysed using cell permeabilization buffer included in the Mem-PER Plus kit, and subsequently solubilized with either the hypotonic solubilization buffer included or solubilization buffer altered to be isotonic. Increasing the salt content Mem-PER Plus Solubilization Buffer increases the extraction efficiency for both Na, K-ATPase and ADP/ATP Translocase 3 proteins

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C= Cytoplasmic Fraction, M= Membrane Fraction, P= Insoluble Fraction

Na,K ATPase

ADP/ATP Translocase 3

Hypotonic Isotonic

43% 71%

74% 83%

C M P C M P