1. No category

January 18, 2012
Sample preparation & protein
enrichment for proteomics and
mass spectrometry
Helen Kim
934-3880
[email protected]
Dept of Pharmacology and Toxicology
McCallum Building, room 460
http://www.uab.edu/proteomics
Jan 18, 2012
HelenKim/UAB/PharmTox
1
Learning objectives
•  “Smart” proteomics:
–  lowest proteome complexity, highest biological
specificity
•  How do we achieve lowest proteome
complexity:
–  Purify according to intrinsic properties
–  Purify according to biological properties
–  Enrich for subcellular compartments
•  If the chemistry in sample preparation has
been ignored, or hasn’t been dealt with
properly, the most “pure” proteomics sample
will give lousy mass spectra.
Jan 18, 2012
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The basic elements of intact protein proteomics:
(1) separation, (2) processing for MS analysis, (3)
identification and characterization
trypsin
2647.50
1705.16
1838.26
1937.22
1994.24
2137.21
2284.25
2384.27
5000
684.11
10000
1461.00
http://www.matrixscience.com
15000
789.57
MASCOT search engine
Counts
ID of parent polypeptide
1122.79 1165.80
1277.83
1048.72
MALDI-TOF mass spectrometry
0
1000
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HelenKim/UAB/PharmTox
1500
2000
2500
Mass (m/z)
3
3000
Handling proteins and peptides
•  Collecting and storing the sample
–  Use the same type of storage device for all the samples
in a study…quality control!!
–  Some samples are sensitive to freezing
•  Mitochondria and other organelle fractions should be
prepared using fresh tissue
•  Samples especially fluids and organelle preparations
should be placed in same volume aliquots and only thawed
one time to avoid the effects of multiple freeze-thaw cycles
–  Freeze fast (into liquid N2)
•  Buffers such as sodium phosphate can selectively
precipitate while ice and water are in equilibrium (down to
-20oC) - this can lead to a substantial change in pH
•  Similarly, thaw fast rather than slowly, then keep on ice.
(thaw by running between your hands, vs letting it sit on
bench while you eat lunch.
Jan 18, 2012
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Tissue disruption/cell lysis
•  Manual and mechanical homogenization
–  Pestle and mortar, Dounce and PotterElvehjem homogenizers, Waring blender,
Polytron
•  Grinding with beads, sonication and
freeze-thaw
•  Osmotic shock - or not!
•  BugbusterTM for bacteria
•  Detergents: CHAPS, Triton X-100, cholate
and deoxycholate
•  Protease
inhibitors
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Parameters that govern the choice of
protein separation method
• 
• 
• 
• 
Purity of protein
Speed of purification
Quantity of protein
What is the question:most important
–  Discovering a new protein/proteome
–  Identifying protein-protein interactions
–  Identifying potential modifications of known
proteins
Jan 18, 2012
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Properties of polypeptides that enable
separation from each other
•  Intrinsic properties
–  Size—number of amino acids
–  Net Charge
•  Biological/functional properties
– 
– 
– 
– 
Intracellular location
Enzyme activity
Undergoes oligomerization
Undergoes modification
Jan 18, 2012
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Some protein separation methods
are both preparative and analytical
•  2D electrophoresis
–  Resolves polypeptides dissociated from each
other
–  Good for isoform detection/quantitation, PTM
changes detection/quantitation
•  2D blue native electrophoresis:
–  Resolves complexes containing intact proteins
–  Can be quantified by MS, or by blotting for
specific proteins
Jan 18, 2012
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Separating proteins by size
•  For purification: Gel filtration
chromatography
•  Analytic: SDS-PAGE
Jan 18, 2012
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Separating proteins by charge
•  Purification: Ion exchange chromatography
•  Analytic: isoelectric focussing
•  But may use SDS-PAGE to monitor
purification
Jan 18, 2012
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Protein functional properties that
can be basis for purification
•  Oligomerization:
–  Each cytoskeletal component
undergoes reversible oligomerization
from its monomers
•  Intracellular location:
–  Change in
–  Increase in same location
•  Protein-protein interaction
Jan 18, 2012
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Wide dynamic range for proteins in
most cells: rationale for protein
enrichment
•  A gel that is overloaded with respect to the
abundant proteins, may still have only barely
detectable or nondetectable amounts of a low
abundance protein
–  If you can t see it, you don’t know it’s there;
–  Even if software detects and quantitates it, you can t do
MS of it, because there isn’t enough protein.
•  No one stain will detect 9 orders of magnitude
differences in abundance of proteins.
•  In MS experiments, bypassing gels, the greater the
complexity of the peptide mixture, the lower the
chance of detecting very low abundance proteins/
peptides.
Jan 18, 2012
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Use “old” information and “conventional”
approaches like differential centrifugation
to enhance proteomics experiments.
hypotonic
lysis and/or
homogenization
Nucleus (pellet)
1,000xg
supernatant
10,000xg
100,000xg
supernatant
+
membranes,
vesicles
Jan 18, 2012
Mitochondria
(pellet)
cytosol
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13
Analysis of mitochondrial proteins
enhanced by purifying that subproteome
Whole Rat Liver Rat Liver Mitochondria 3 10 3 10 2D gel analysis using the Invitrogen ZOOM system (Courtesy of Shannon Bailey Lab – Whitney Theis and Kelly Andringa) Jan 18, 2012
HelenKim/UAB/PharmTox
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The good news:
Several subcellular proteomes have been
catalogued.
Compartment
# polypeptides in SWISSPROT
as of 2000
Mitochondria (1000/cell)
269------5% of total
Lysosome (400/cell)
50------1% of total
Peroxisome
35------0.6%
ER and Golgi apparatus
157------3%
Nuclei (5% cell volume)
964-----17%
Others (cytosol, membrane)
4228----75%
total: 5703
(Jung et al. [2000] Electrophoresis)
Note date: this is old!!! But the principle is the same;
homework for class, find current numbers, please.
Jan 18, 2012
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Antibodies can reduce the complexity of
the proteome, as well as enhance biological
specificity, by 10,000-fold
A cell lysate: 6,739 polypeptides
An immune complex of 1-10 polypeptides
(why might there be more than one
polypeptide?)
Which sample would you rather deal with on a 1D
or 2D gel?
Jan 18, 2012
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2D Western blot + Mass spectrometry identified
colitogenic antigens from a particular bacterium
Stained 2D gel of one lysate
Western Blot
A B C
D E
F G
H I J K L
M
N!
Bacterial lysates probed with mouse serum
Duplicate 2D gel Western blotted with the same serum
MS
Jan 18, 2012
gi|29338205
Mass: 45415
MOWSE Score: 100
Putative identification:
tetratricopeptide repeat family
protein [Bacteroides
thetaiotaomicron VPI-5482]
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(Elson et al., unpublished data)
2D DIGE of immunoprecipitates to
determine tubulin-binding proteins
A
B
Overlay of A and B images
GREEN (A): starting tubulin and MAPs
RED (B): antibody alone, 8 ug
Conclusion:
tubulin, MAPs
and antibody resolved from
Jan 18, 2012
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each other, without having to deplete of antibody
Complexity in initial 2D analysis of tubulin
immunoprecipitates: Something in the MAPs coprecipitates with anti-tubulin
A
B
A and B overlay
GREEN (A): pellet of anti-tubulin + MAPs, no tubulin
(commercial preparation);
RED (B): pellet of anti-tubulin + tubulin, no MAPs;
YELLOW:
in both pellets;
IDEALLY,
Jan 18, 2012
proteins common
HelenKim/UAB/PharmTox
there should be no green
19
Affinity purification: Antibodies are
available for many protein
posttranslational modifications
•  Phosphorylation
•  Glycosylation
•  Oxidative modifications:
–  Protein carbonyls
–  Reactive aldehyde adduct formation: 4HNE
•  Keep in mind that these modifications
each involve mass changes, thus can
be detected directly by MS.
Jan 18, 2012
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Structures important in protein electrophoresis
to know
2
1
4
3
6
7
5
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The action of a “reductant”
A disulfide bond
“the reductant”
(believe it or not, from Wikipedia, 2011)
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The interplay of protein structure and SDS: Single
mutations in a hairpin sequence affect mobility of
an intact polypeptide on SDS-PAGE.
(Taken from Rath et al., PNAS 2009)
Jan 18, 2012
©2009 by National Academy of Sciences
HelenKim/UAB/PharmTox
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Relationship between hairpin conformation and
detergent binding.
(Taken from Rath et al., PNAS 2009)
Jan 18, 2012
©2009 by National Academy of Sciences
HelenKim/UAB/PharmTox
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Correlation of SDS binding and hydropathy.
(Taken from Rath et al., PNAS 2009)
Jan 18, 2012
©2009 by National Academy of Sciences
HelenKim/UAB/PharmTox
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SDS-binding and mobility on SDS gels.
(Taken from Rath et al., PNAS 2009)
Jan 18, 2012
©2009 by National Academy of Sciences
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Why do we care about SDS binding and
minute differences in protein structure:
•  Western blot analysis can detect a polypeptide at an
unexpected mw; doesn’t mean its mass is different
necessarily in the mutant or diseased tissue;
•  Western blot detection of multiple bands may reflect
structural or conformational heterogeneity in that one
protein in the sample, not cross-reactivity with other
proteins.
•  Cannot presume a polypeptide will be at or near its
predicted or WT mw.
Jan 18, 2012
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27
Wide dynamic range for proteins in
most cells: rationale for protein
enrichment
•  A gel that is overloaded with respect to the
abundant proteins, may still have only barely
detectable or nondetectable amounts of a low
abundance protein
–  If you can t see it, you don’t know it’s there;
–  Even if software detects and quantitates it, you can t do
MS of it, because there isn’t enough protein.
•  No one stain will detect 9 orders of magnitude
differences in abundance of proteins.
•  In MS experiments, bypassing gels, the greater the
complexity of the peptide mixture, the lower the
chance of detecting very low abundance proteins/
peptides.
Jan 18, 2012
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28
Benefit of removing salt from tryptic digest
1929.87
100
90
1993.962209.97
80
% Intensity
8477.0
1868.97
70
60
2148.00
2897.04
1804.90
Salt Contamination
2225.00
50
40
30
20
10
0900
1520
2140
2760
Mass (m/z)
1804.96
100
Trypsin autolysis peak
% Intensity
90
80
30
20
2148.00
1475.78
Jan 18, 2012
ZipTip (desalting)
1928.96
1868.94
2290.10
1439.74
2383.97
1179.65
2706.12
2502.28
10
0 899.0
2.7E+4
1994.97
60
40
0
4000
2164.04
70
50
3380
1519.2
2139.4
2759.6
Mass (m/z)
3024.06
3379.8
HelenKim/UAB/PharmTox
0
4000.0
29
Preparing proteins for peptide mass
fingerprinting (1)
•  The mass spectrometry procedure has to be
preceded by high resolution protein
chromatography steps……the LC part of LC-MS.
•  Or, it can be a sample with a few intense bands of
interest - e.g., a recombinantly expressed protein in
bacteria lysate.
•  SDS-PAGE may be the best thing you can do for
your protein preparation…it gets rid of lots of
potential contaminants, and at the same time
concentrates the proteins into “bands.”
Jan 18, 2012
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Preparing proteins for peptide mass
fingerprinting (2)
•  Once the protein is precipitated in the SDS gel
matrix, electrolytes/salts and the SDS are largely
removed by washing the gel pieces with 50%
aqueous acetonitrile containing 25 mM NH4HCO3
buffer, pH 8
•  The gel is dehydrated and rehydrated in 25 mM
NH4HCO3 buffer, pH 8 to which trypsin added for
overnight digestion.
•  The resulting peptides are extracted with 50%
aqueous acetonitrile containing 25 mM NH4HCO3
buffer, pH 8 - the extract is evaporated, and
brought up in MS-compatible buffer/solvent.
Jan 18, 2012
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Activity-based protein profiling
(ABPP) to increase biological
specificity of proteomic anslysis
Jan 18, 2012
(Taken from Bogyo, PNAS, Feb, 2010)
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32
Tandem orthogonal proteolysis-activitybased protein profiling (TOP-ABPP):
reducing complexity and increasing
biological specificity at the same time
CC reagents:
Click chemistry is
at the heart of this
(Weerapana, Speers, Cravatt, Nature Protocols,
Jan 18, 2012
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Take home messages regarding protein
purification
•  Reduce complexity of proteome
–  by conventional protein separation approaches (size
exclusion, ion exchange, reverse phase).
–  Let biology work for you
•  Enrich for subcellular compartment
•  Affinity chromatography indicated by response to ligand
•  Oligomerization, protein-protein interactions
•  Choice of purification/separation approach
governed by
– 
– 
– 
– 
Abundance of sample
Abundance (if known) of protein in question
Question being asked
What technologies you can access readily
•  LAST BUT NOT LEAST: What you can afford
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Take home points, part II
• 
• 
• 
• 
• 
• 
What proteomics technology gives back is like any other
analytical approach: the quality is as good as what you put in;
Be mindful of distinguishing between low abundance proteins
versus low level contaminants;
Keep in mind conventional approaches like Western blotting
to validate proteomic results;
Purify, purify, purify before running any proteomic experiment.
Formulating a hypothesis forces you to incorporate biological
information that can enhance proteomic analysis.
The “end result” in proteomics is just a beginning:
I. 
II. 
III. 
Some changes are causal to the disease/phenotype;
Some are real but not causal;
Some could be response of the cells/tissues TO the disease, not
causing the disease.
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Suggested readings
•  Cañas et al., 2007. Trends in sample preparation for
classical and second generation proteomics Journal of
Chromatography A, vol. 1153, pp 235-258.
•  Ishihama Y, Rappsilber J, Mann M. Modular stop and go
extraction tips with stacked disks for parallel and
multidimensional peptide fractionation in proteomics. J
Proteome Res. 2006 Apr;5(4):988-94.
•  Durbin KR, Tran JC, Zamdborg L, Sweet SMM, Catherman
AD, Lee JE, Li M, Kellie JF, Kelleher NL, Intact mass
detection, interpretation, and visualization to automate TopDown proteomics on a large scale. PROTEOMICS Special
Issue: Focus on Top-Down Proteomics 10 (20): 3589–3597,
2010.
2012
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