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Membrane Specific Proteomics
Martin R. Larsen
Protein Research Group
Dept. of Biochemistry and Molecular Biology
University of Southern Denmark
Odense, Denmark
E-mail: [email protected]
Membrane proteins make up at least one third of total proteins in the cell
Nucleus
Endoplasmic Reticulum
Cell membrane
Vacuole
Mitochondrion
Peroxisomes
Lysosomes
Golgi
Secretory vesicles
Membrane proteins
Function:
- Inter-/intra-cellular communication
- Cellular attachment
- Maintenance of the cell membrane potential
- Mediation of the transport of ions and proteins
- Regulation of vesicle transport
- etc…….
Transmembrane proteins
(1-13 membrane spanning regions)
Peripheral proteins
(Loosely attached to the membrane
e.g., via lipid anchors)
Because of the amphipathic structures, membrane proteins
are notoriously difficult to handle by any technique
Proteomic´s strategies
1. Traditional (European)
Protein mixture
3. GeLC-MS
Protein mixture
SDS-PAGE
2 D PAGE
Band excision
Differential display
Liquid chromatography
Mass spectrometry
Spot excision
2. Modern (American)
Protein mixture
Proteolytic digestion
Liquid chromatography
Mass spectrometry
Peptide fragmentation
Peptide fragmentation
Protein identification by
MS or MSMS
Database searching
Database searching
The big question:
or
”2D or not 2D”
“LC or not LC”
Protein solubilization for 2DE
Highest resolution obtained with 2-DE is under denaturing conditions!
Chaotropes (protein unfolding/denaturation)
- Urea (5 - 9.5 M)
- Thiourea (2 M)
Surfactants
- CHAPS
- NP-40
- Triton X-100
- Sulphobetaines (e.g. SB 3-10,
ASB14, C8Ø)
Reducing agents
- DTT
- DTE
- Tributyl phosphine (TBP)
Bonds/forces within a protein
or between proteins
Non-covalent:
-Hydrogen bonds
-Ionic bonds
-Hydrophobic interaction
-Van der Waals forces
Covalent:
-Disulfide bonds (C-C)
Santoni V., electrophoresis 2000
Molloy M., electrophoresis 2000
Use of different IEF buffer conditions to fractionate proteins with
increasing hydrophobicity.
Reagent 1: 40 mM Tris
Reagent 2: 8 M urea, 4% CHAPS,
40 mM Tris, 0.2% Bio-Lyte
3/10 ampholyte, 2 mM TBP
Reagent 3: 5 M urea, 2 M thiourea,
2% CHAPS, 2% SB 3–10,
40 mM Tris, and 0.2% Bio-Lyte
3/10 ampholyte
Stronger detergents e.g. ASB14
Or SDS-PAGE
A: Normal sample solution
B-D: BioRad sequential
extraction kit (extraction 1-3)
Simple protocols for purifying membranes/membrane proteins
prior to IEF solubilization
Triton X100 stripping:
Strip cytosolic proteins from the plasma membrane (PM)
Triton X100
Supernatant (soluble proteins)
Pellet (membrane proteins)
Centrifugation
Triton X114 (phase partitioning)
Triton X114
Upper phase – soluble proteins
Centrifugation
lower phase – insoluble proteins (detergent phase)
Carbonate treatment: Closed vesicles are converted to open membrane sheets, and content proteins
and loosely attached proteins are released in soluble form.
Sample sonicated
in 40 mM Tris
(pH 11)
Supernatant (soluble proteins)
Sodium carbonate
Incubation on ice
(10  excess)
Centrifugation
(90 min at 15000 g)
Pellet (membrane proteins)
Sodium carbonate
TX114 Lower phase
TX114 Upper phase
Insoluble in TX100
Soluble in TX100
Total membrane
Cells treated with the different protocols
Followed by SDS PAGE and immunoblotting
Santoni V, Kieffer S, Desclaux D, Masson F, Rabilloud T.
Electrophoresis 2000 Oct;21(16):3329-44
Different zwitterionic surfactants (”detergents”) used
in 2D membrane proteomics.
Conventional 2D electrophoresis
BioRad reagens 3
Calbiochem
Calbiochem
Insoluble fraction from TX100
Santoni V, Kieffer S, Desclaux D, Masson F,
Rabilloud T.
Electrophoresis 2000 Oct;21(16):3329-44
Sample taken from the
sodium carbonate extraction
Santoni V, Kieffer S, Desclaux D, Masson F, Rabilloud T.
Electrophoresis 2000 Oct;21(16):3329-44
Proteome analysis of the Pseudomonas aeruginosa, PAO1,
Membrane proteome - Nouwens AS et al., Electrophoresis 2000 Nov;21(17):3797-809
Cell pellet
Lysis in 40 mM Tris buffer, pH 11
Incubation in 0.1 M ice-cold
sodium carbonate
Centrifugation at 15000 g
Wash pellet in 40 mM Tris
buffer, pH 11
Solubilization in sample solution
Conventional sample solution: 7M Urea, 2M
Thiourea, 2% CHAPS, 2% SB 3-10, 2mM TBP,
0,5% carrier ampholytes
Optimized sample solution: 7M Urea, 2M
Thiourea, 2% CHAPS, 2% SB 3-10
1% ASB 14, 2mM TBP, 0,5% carrier ampholytes
Protein identification
Based on MALDI MS:
Number of previously
characterized proteins:
70
Number of proteins which
have significant homology
to known membrane proteins
from other organisms:
88
”New” proteins unique
To Pseudomonas aeruginosa,
PAO1:
30
Approximately 300-350 proteins could
be resolved on the gel using Coomassie blue
220 spots were excised form the gel for MS analysis
Differences between normal and optimized sample solubilization
2
1
1. Outer membrane porin GRAVY –0,418
2. Hydroxamate-type ferrisiderophore receptor GRAVY –0,574
GRAWY: Grand average hydropathy value (a measure of the overall protein hydrophobicity)
The higher the GRAVY value the more hydrophobic is the protein !!
The ability to solubilize a protein does NOT depend on the
overall hydrophobicity of the protein – it is more likely that
specific regions within the protein effect the solubilization!!
Characterization of the membrane proteome from sorted X- and
Y- Chromosome bearing sperm cells
Background: Currently, the X and Y chromosome-bearing sperm that determines
sex at fertilization as female and male, respectively, is sorted by using a
modified flow cytometer. The method is relatively inefficient for sorting high
amounts of sperm cells, resulting in an insufficient number of the sperm cells
being successfully sexed.
Aim: Localization and identification of putative sex-specific membrane proteins
which could result in the development of new more efficient methods to isolate
X and Y chromosome-bearing sperm cells which are of key interest for livestock
producers by enabling them to choose the sex of offspring.
Optimerization of IEF sample buffer
3  108 unsorted pig sperm cells
Sodium carbonate extraction
7M Urea, 2M Thiourea, 1% ASB 14,
2mM TBP, 0,5% carrier ampholytes
pH 4
solubilization
7M Urea, 2M Thiourea, 2% CHAPS, 2%
SB 3-10, 1% ASB 14, 2mM TBP, 0,5%
carrier ampholytes
pH 7
pH 4
70 KDa
10 KDa
pH 7
Optimization of the Na2CO3 extraction with respect to starting material
3  10
Marker proteins
8
–
cells
membrane associated
Indicate cytoplasmic proteins !
6  10
8
cells
Cell sorting
Pig sperm X and Y, Chromosomal difference: 3.5 %
R1
Dead cells
Sorted Pig sperm
Pig Y
Pig X
5 4
6
11
7
9
8
10
3
2
1
Marked spots differ in expression with > 1.7
22 Proteins showed expression changes > 1.7
- 11 of those could be identified by MALDI tandem mass spectrometry
Spot
number
Protein identification
Accession
number
Location
Comments
1
Ras-related protein Rab-2A
Q01971
Endoplasmic reticulum
Lipid anchor
2
Ras-related protein Rab-2A
Q01971
Endoplasmic reticulum
Lipid anchor
3
Glutathione S-transferase, mu 5
P46439
Cytoplasmic ?
4
ATP synthase alpha chain
P19483
Mitochondrial inner
membrane
5
ATP synthase alpha chain
P19483
Mitochondrial inner
membrane
6
ATP synthase beta chain
P00829
Mitochondrial
7
ATP synthase beta chain
P00829
Mitochondrial
8
Glutathione S-transferase, mu 5
P46439
Cytoplasmic ?
9
Voltage-dependent anion-selective channel
protein 2
P45880
Outer mitochondrial
membrane
10
Voltage-dependent anion-selective channel
protein 3
Q9Y277
Outer mitochondrial
membrane
11
Outer dense fiber 2 protein
O35496
Sperm tail membrane protein
Considering 95 % purity in the cell sorting no differences could be
detected on the 2 D gels, which could provide basis for
discrimination between X and Y in the cell sorting.
Overview of the 2 D gel experiment
KDa
Total number of proteins detected on the gel.
300
Total number of protein spots tried identified from the 2
D gel.
118
Number of spot where the protein is identified.
52
Number of protein spots on the gel with ID protein
located to the mitochondria.
35
Number of protein spots on the gel with ID protein
located to the sperm tail.
12
Number of protein spots on the gel with ID protein
located to the endoplasmatic reticulum.
5
90
30
3
pH
10
The majority of the proteins identified using the 2DE
approach were membrane proteins derived from either
the mitochondrial membrane or the sperm tail.
Shaving membranes
Whole Cell
Limited
proteolysis
LC-MSMS
Enriched Membrane
sample
LC-MSMS
Christine C. Wu and John R. Yates, III. Nature biotechnology 21, 262-267
Liquid chromatography tandem mass spectrometry – membrane shaving
Limited tryptic proteolysis
of sorted cells
100
Reversed-phase capillary LC-MS
2
3: TOF MSMS ES+
%
0
100
2: TOF MSMS ES+
1
%
0
100
1: TOF MS ES+
%
0
40.00
50.00
60.00
70.00
Time
80.00
90.00
100.00
110.00
Overviews of the LC-MS analysis
MS-Tag
Score
Database
Accession
Protein Name
Located to
the surface
90.97
17388906
Cenexin 2
59.00
897763
Beta-tubulin
48.42
14194458
A-kinase anchor protein 3
47.79
5791527
Semenogelin I protein (SGI)
38.19
106185
GTP-binding protein Rab2
33.08
27713216
Tubulin alpha-3/alpha-7 chain
28.75
21493039
A kinase (PRKA) anchor protein 4 isoform
2

19.98
2253123
75 kDa fibrous sheath protein

17.87
497832
zona-pellucida-binding protein (sp38)

Number of proteins identified
using LC-MS/MS.
34

Number of known proteins
identified by LC-MS/MS.
17

Number of hypothetical proteins
identified by LC-MS/MS.
17
Number of identified known
proteins found to be associated
with the surface of the sperm
cells.
10
(59%)
Number of fragment spectra
generated
1500
Number of ”peptide” fragment
spectra.
497

16.59
20381082
LanC-like protein 1

16.30
27500209
Glypican-5 precursor

15.03
10305336
peroxiredoxin 5
14.86
8453169
Sperm-associated antigen 4 protein
14.83
1834491
EYA3
14.54
609541
Glucocorticoid-regulated endocrine
protein
14.36
13507710
ropporin
14.33
4099365
glutathione-S-transferase, mu 5


The two techniques are highly complementary
2 DE
Advantages
• Separation of protein isoforms (modifications)
• More reliable protein identifications!
• High sequence coverage
Disadvantages
• Poor detection of low abundant proteins
• Poor detection of basic proteins
•Need more material than LC-MSMS
• Quantitation can be relatively hard
LC-MSMS
Advantages
• Sensitive (below pmol level)
• Identification of ”difficult” proteins
• Quantitation possible – e.g. ICAT/Stable isotope
labeling
Disadvantages
• Large amount of data
• Difficult to detect post-translational
modifications
• Impossible to detect mutations
• The majority of proteins are identified based
on only one single peptide.
Overall the two techniques complements each other
Example: Diabetes type 1
Diabetes Mellitus (DM) is a group of disorders characterized by chronic
hyperglycemia with disturbances of carbohydrate, fat and protein metabolism
resulting from defects in insulin secretion, insulin action or both.
T1D: Absolute insulin deficiency
due to an autoimmune associated
destruction of the insulin
producing ß-cells in the islets of
Langerhans.
T2D: Relative insulin deficiency
due to decreased effect of insulin
in the target tissues e.g. muscles
and adipose tissue (insulin
resistance) or due to a secretory
defect of insulin with or without
insulin resistance.
Pancreas
ß-celle
Muskelfiber
Blodkar
Insulin
Glucose
Copenhagen Model
IL-1 ?
Virus ?
Chemicals ?
Nutrition ?
•
•
• •
•
•
•
•
• •
• •
•
IL-1
•
•
•
•
TNF a
• •
•
Beta
cells
Klonal Tand Blymfocyt
ekspansion
Mø
+
NO
•
Nerup, J. Et al., 1994, Diabetologia 37
(Suppl 2), S82-S89.
•
O 2•
•
•
•
•
•
•
•
•
IFN g
IL-1
Mø / EC
Th
lymphocyte
O 2-
NO
Cytokines => inducible nitric oxide synthase expression => NO generation
Investigation of the effect of Interleukin-1 on isolated islets
Islet isolation
Cytokines => iNOS expression => NO generation => -cell
destruction
L-Arginine
analogs (NMMA)
S35-methionine
IL-1ß + S35-methionine
SDS PAGE
Mass spectrometric
protein identification
H. U. Andersen et al, Electrophoresis 1997
P. Mose Larsen et al, Diabetes, 2001
pH
The effect of interleukine 1 on the plasma membrane sub-proteome of
insulin producing beta cells
Plasma
membrane
-Plasma membrane proteins constitute only 3-5 % of
the whole proteome.
-Cell-cell communication/attachment
-Signalling.
-Transport of ions and proteins.
-First barrier to the environment
-Important potential drug targets and markers for the
pharmaceutical industry
Important in Diabetes
Plasma membrane sub-proteome - strategy
SILAC: Stable isotop labeling with isotop amino acids
Cell line in normal
amino acid media
Cell line in media
containing Lys(13C) +
Arg(13C,15N)
 IL - 1
+ IL - 1
Reference
Experiment 1
Pool 1:1
Cell lysis - mechanical in succrose buffer
Trypsin digestion
TiO2 purification
Flowthrough
Bound - phosphopeptides
Succrose centrifugation and Na2CO3 treatment
LC-MSMS
Centrifugation
Solubilization using SDS sample buffer
SDS - PAGE
LC-MSMS
Tumor-associated calcium
signal transducer 1 [Rattus norvegicus]
Plasma membranes
In-solution trypsin digestion
EMGEIHR
PM fraction SILAC, Trp, SCX, 10mM KCL
u04963MRL 1654 (34.497) Sm (SG, 3x3.00); Cm (1652:1655)
Strong cation exchange
LC-MSMS
1: TOF MS ES+
148
436.26
100
5 Da
%
441.26
436.76
441.77
437.26
443.28
434.26
434.77
435.31
438.27
439.28
440.78
442.28
439.79
443.78
444.79
442.78
435.78
0
m/z
434
435
436
437
438
439
440
441
442
443
444
10 mM KCL fraction
GESLFHSSK
PM fraction SILAC, Trp, SCX, 10mM KCL
u04963MRL 1681 (38.088) Cm (1679:1682)
1: TOF MS ES+
135
496.30
100
3 Da
496.32
499.30
496.28
495.33
496.81
%
494.33
497.31
495.83
494.83
495.76
494.61
501.32
499.80
497.84
497.38
496.39
498.32
501.83
500.30
498.82
500.81
0
m/z
495
496
497
498
499
500
501
Cation-independent
mannose-6-phosphate receptor
LVSFHDDSDEDLLHI.-
L+H
C-terminal peptide from protein
– can not be quantified by SILAC
Golgi vesicular membrane trafficking
protein p18
SLSIEIGHEVK
L
H
Glycosyl-phosphatidylinositol anchored proteins:
Membrane attached surface proteins
GPI-anchored
protein
C terminus
Man
Ins
Man
P
Acts as
• enzymes
• adhesion molecules
• receptors
• antigens
•…
P
DAG
Interior
Man
Widespread class of
membrane proteins
Plasma membrane
Exterior
GlcN
Ethanolamine
NH2
Ferguson et al. (1988) Science 239, 753-759
Strategy for isolation and identification of
GPI-anchored proteins
Cell culture
Isolation of membrane fraction
Isolation of GPI proteins
•PI-PLC treatment
•Two-phase separation
PI-PLC
SDS-PAGE of extracted protein
LC-MSMS
Identification of GPI-proteins by
nanoLC-MS/MS
Elortza, F et al, 2004
Identification of proteins in human lipid rafts
10 slices
LC-MS/MS
DB search
16 proteins
Trypsin
Literature
Sequence
analysis
6 GPI-anchored
proteins
Human GPI-proteins (lipid rafts)
Accession no.
• Alkaline phosphatase
P05186
• Decay acceleration factor
P08174
• Folate receptor 1
P15328
• CD59 glycoprotein
P13987
• Carboxypeptidase
P14284
• Urokinase plasminogen activator receptor
Q03405
# peptides
11
9
8
3
2
1
OTHER: Fibronectin receptor-CD29 (P05556), Galactoprotein-CD49c (P26006),
Melanoma adhesion molecule-CD146 (P431214), F2 heavy chain antigen-CD98 (P08195), EpicanCD44 (P16070), 78 kDa glucose-regulated protein (P11021), Mesotheline/megakaryocyte
potentiation factor (Q9UK57), Actin /g actin (P02571/P02572), Voltage dependent anion channel
(P21796), B-cell antigen receptor complex associated protein alpha-chain-CD79 (P11912).
GPI-proteins in A. thaliana

Genome: ~ 25.000 ORFs

Prediction: 210 GPI-APs (Borner et al., 2002)

Only few GPI-APs have been experimentally
verified

Good test case for our strategy
Collaborators: T. Nühse and Scott Peck, Norwich, UK.
Isolation of GPI-anchored proteins from A. thaliana cell line
B
A
100
PI-PLC
Band #
+
-
M
kDa
116
1
2
3
4
5
6
7
8
9
10
11
12
%
0
32
36
40
97
66
44
48
Tim e
(min)
52
C
100
45
MS
660.96
%
31
13
14
15
BPI
0
900
700
500
1100
D
21
R
16
D
L
S
I
E
S
D
G D
D
V
y4
100
y5
y1
14
m/z
1300
%
y2
MS/MS
y7
y9
y6
y3
y10
y8
y11
0
100
300
500
700
900
1100
1300
m/z
Identification of GPI-anchored proteins in A. thaliana by LCMS/MS (Q-TOF)

64 protein identified in 16 gel slices:

44 bona fide GPI-anchored proteins validated by
sequence analysis and prediction tools

20 proteins with TM domains or secretion signals,
i.e. non-GPI-APs
A few other ways to look at membrane proteins

Top-down proteomics. HPLC separation of membrane proteins in high
concentration of formic acid. Has to use FT-MS instrumentation

Labeling of the plasma membranes using biotin derivatives that are not
membrane permeable. Problem: the biotin derivatives are almost always
permeable leading to purification of non-membrane proteins.

We are working on alternative methods for purification of especially
plasma membrane proteins………..
Conclusion

Membrane proteins can be efficiently separated by 2 DE using
strong detergents in the IEF buffer.

Sodium carbonate treatment or high salt treatment is crucial for
elimination of non-membrane proteins – cytoplasmic
contaminants…

Surface membrane proteins can be efficiently identified using
LC-MSMS of peptides derived from proteolysis of intact cells or
enriched membrane fractions. However, cell sorting is crucial
here….

2DE and LC-MSMS is highly complementary for any proteomic
work.
Acknowledgements
University of Southern Denmark
Peter Roepstorff
Ole N. Jensen
Felix Elortza
Australian Proteome Analysis Facility
Stuart Cordwell
Brad Walsh
Derek Van Dyk
Amanda Nouwens
Faculty of Veterinary Science
Gareth Evans
Bengt Eriksson