1. Overview
Purpose: 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.
Introduction
Membrane 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
References
1. 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
-80o
C 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 (125µg) was
immunoprecipitated with Thermo Scientific Anti-AT1B1 antibody (5µg) and Thermo
Scientific Pierce Protein A/G Magnetic Beads using MS compatible wash and elution
buffers. Eluted samples were dried down, and stored at 4o
C.
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.
.
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 (50µg) 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
.
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.
.
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).
.
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
.
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