Download Sample Preparation I (Protein Purification)

Proteomics
GET THE BIOLOGICAL ANSWERS YOU WANT
SAMPLE PREP AND SEPARATIONS
Cindy L. James, PhD
Medical Biochemist/Protein Chemist
Our life is maintained by molecular network systems
(From ExPASy Biochemical Pathways; http://www.expasy.org/cgi-bin/show_thumbnails.pl?2)
Protein
interactions are at
the core of the
entire system of
any living cell…
…AN ABSOLUTE
REQUIREMENT TO
UNDERSTAND!
Same genome,
different proteome
Only 21,000 genes to encode a half million proteins!
PROTEINS
RNA
DNA
IE: Alternative splicing - In humans, many genes contain
multiple introns and make multiple proteins!!
intron 1
1
intron 3
intron 2
2
3
1
2
3
intron 4
4
4
5
5
Usually all introns must be removed before the mRNA can
be translated to produce protein
Be careful of genetic knock-outs! You may have disrupted five other pathways with
the target pathway!
Proteins come in many
sizes, shapes and
chemistries
Hydrophobic, lipid sticks
to it, will show in lysis
pellet
Phospholipid bylayer
Hydrophillic, can
shear and show in
supernatant
Binds and shows with
RNA, Reflects higher
molecular weight,
Very Positively charged
What do you want to know?
◦ Which proteins are present? In what isoforms?
◦ What post-translational modifications?
◦ In what concentrations (quantification)?
◦ What “signature” does protein have that will relate to other
pathogenic or cancer related proteins
◦ What pathways and involved proteins will assist in
determination of drug therapy
◦ WHO DOES IT ‘PLAY’ WITH?
Today, we largely address these questions via mass
spectrometry, but
◦ GOOD SAMPLE PREPARATION IS ESSENTIAL!!
Not Every Sample or Cell-line has the
same proteome!
Our samples come from:
Bacteria
Feces
Food
Hair
Plants
Roots
Sediment
Seeds
Sludge
Tree
Tissue
Urine
Nasal secretion
Yeast
Blood
Plasma
Saliva
Urine
For Example: Tissue samples obtained from a biopsy, or
Biopsy
during surgical removal of a tumor can be used to classify
the type of tumor found in the patient!
Not all Proteins are produced by the cell
in equal amounts!
Favored research drug targets (signal proteins) are
actually low in abundance!
◦ kinases,
◦ proteases
◦ hydrolases of all sorts
◦ receptors (most likely)
◦ Researcher needs to aim for regulatory choke point
and bottleneck proteins for targets
Some Experiment Challenges
Statistics! Analyses = difficult to duplicate
◦Ie: Statistically better = grow many plates at once and harvest all
at once, not many different growths. Proteome will be more equal
between samples
Difficult to prepare pure samples
Cellular protein expression very sensitive to environmental
conditions AND pH’s
Gel work may not run identically from time to time
Metabolite Sample Extraction can also be tricky
Choosing an extraction method
• No universal extraction method exists
• Some
• Do
solvents may degrade certain compounds
you know the characteristics of metabolites you want to
extract?
Notes on Sample Preparation
Contaminants (nucleic acids, lipids, and carbohydrates) can
cause problems
There is no single protocol for “cleaning up” the protein
sample!
Researchers combine procedures to reduce unwanted
components.
Things to ask before sample prep and why
What are you looking for?
◦ Are they fishing for lots of global proteins, or looking for one
precious gem?
Where do you expect to find it?
◦ Do they think the protein is nuclear? Cytosolic? Expressed out of
the cell?
Does your protein have any characteristics that make it
different from all others?
◦ Does it bind DNA? Ligands? ATP?
Is your protein hydrophobic? Bind lipids? Sugars?
What is the protein’s PI? (isoelectric point)
Has anyone published on this or something similar?
OPEN YOUR CELLS AND TISSUES
Does your lysis buffer make a difference?
YES!!!
Cells and tissues need to be lysed to release the proteins of interest.
Lysis buffers differ in ability to solubilize proteins.
SDS and other ionic detergents are harshest but give the highest yield
Are you doing IP’s and WB’s?
ANTIBODIES:
- recognize reduced and denatured protein…use these conditions first.
- Some antibodies will only recognize a protein in native, non-denatured form - don’t use
denaturing detergent (SDS, deoxycholate, and somewhat less denaturing, Triton X-100).
IP FOR PROTEIN-PROTEIN STUDIES:
- Use non-ionic IGEPAL CA-630, less denaturing and better for kinase activity and protein interactions
Do not use RIPA if looking for protein:protein interactions.
Are you looking for phosphorylation of proteins, protein-protein interactions, or membrane
bound
Use Sulfo-betanes, IGEPAL CA-630 Buffer – for cytoplasmic, membrane-bound, or whole cell extracts. If
protein of interest isn’t completely extracted from insoluble material or aggregates, use harsher
ionic detergents that assist proteins into solution.
Are you interested in total protein levels of a protein
Try RIPA. RIPA buffer can give lower background in immunoprecipitation, but can denature some proteins.
Surfactant should MIMIC the area around your protein!
IGEPAL-CA360
CHAPS
Polar head
groups
Phobic tails
Amino acids are separated due to cation exchange interactions at the right pH.
Chaotropic ions like urea interact nicely with the ammonium groups of sulfobetaine
polymers to dissolve aggregate protein.
ASB-14
PHOSPHOLIPIDS
SDS
Protein location
Whole Cell
Cytoplasmic (soluble)
Cytoplasmic (cytoskeletal bound)
Membrane bound
Nuclear
Mitochondria
Buffer recommended
RIPA
Tris-HCl
Tris-Triton
CJ’s MemLysis or RIPA
use nuclear fraction protocol and RIPA
use mitochondrial fraction protocol, high salt & IGEPAL
Enrich for your elusive target protein by fractionation.
Can load more of the desired protein per gel lane. Will help removal of potentially
cross-reactive proteins from unused fractions.
FOR MEMBRANE PROTEINS
CJ’S MemLysis Buffer – used for signal and membrane proteins. A
chaotrope plus aminosulfobetaine to compete with lipid/hydrophobic amino acid
that involve imbedded membrane proteins.
Common Separation Techniques
Separation by Subfraction
GET YOUR PROTEINS AWAY FROM ALL THE
OTHER KIDS IN THE PLAYGROUND!
Differential Centrifugation
Zonal centrifugation
Summary of initial steps of protein
purification
•
•
•
•
Choose source of proteins.
Solubilize proteins.
Stabilize proteins.
Specific assay for protein of interest
– Enzymatic activity, immunological activity, physical
characteristics (e.g. molecular mass, spectroscopic properties,
etc.), biological activity
• Assay should be:
–
–
–
–
Specific
Rapid
Sensitive
Quantitative
Taking Advantage of the
Isoelectric Point
Know the pI of your proteins of interest!
Every protein has a pI (point where there is an
OVERALL ZERO charge, not where there are no
charges on your protein!)
INDUSTRY RULE:
If your buffer is at the pI of the protein(s) you are
after, you WILL lose your protein in precipitate!
Isoelectric Points of Several Common Proteins
Protein
Pepsin
Ovalbumin (hen)
Serum albumin (human)
Tropomyosin
Insulin (bovine)
Fibrinogen (human)
g-Globulin (human)
Collagen
Myoglobin (horse)
Hemoglobin (human)
Ribonuclease A (bovine)
Cytochrome c (horse)
Histone (bovine)
Lysozyme (hen)
Salmine (salmon)
pI
1.0
4.6
4.9
5.1
5.4
5.8
6.6
6.6
7.0
7.1
9.4
10.6
10.8
11.0
12.1
Using pI to separate your protein(s)
What buffer would you use? Which pH range is best?
[H+]
pI ~5
Protein becomes increasingly +chg
PROTEIN B
----------------------------------
PROTEIN A
Buffer pH
7.5
[OH-]
Protein becomes increasingly -chg
pI ~8.5
Salting Out Your Protein
• Solubilized proteins can be purified based on overall charge,
ionic strength, polarity
• Ammonium sulfate (NH4SO4) commonly used to “salt out”
• Takes away water making protein less soluble because
hydrophobic interactions increase
• Different aliquots taken as function of salt concentration to get
closer to desired protein sample of interest (30, 40, 50, 75%
increments)
• One fraction has protein of interest
Solubility of caboxy-hemoglobin at its isoelectric
point as a function of ionic strength and ion
type.
Solubility of proteins
• Water-miscible organic solvents also precipitate proteins.
– Acetone, ethanol
– Low dielectric constants lower the solvating power of their
aqueous solutions for dissolved ions.
• This technique is done at low temperatures (0 ºC) because at higher
temperatures, the solvent evaporates.
• Can magnify the differences in salting out procedures.
• Some water-miscible organic solvents (DMF, DMSO) are good at s
• Solubilizing proteins (high dielectric constants).
Certain ions (I-, ClO4-, SCN-, Li+, Mg2+, Ca2+ ) increase the solubility
of proteins rather than salting out!
Solubility of proteins
• A protein in a pH near its isolectric point is not
subject to salting in.
• As the pH is moved away from the pI of the
protein, the protein’s net charge increases and it
is easier to salt in.
• Salts inhibit interactions between neighboring
molecules in the protein that promote
aggregation and precipitation.
• pI’s of proteins can be used to precipitate
proteins.
Separating by Column Chromatography
Column Chromatography
Ion exchange chromatography – separation by charge
Beads have charged group:
+ charge binds acidic amino acids - anionic chromatography
- charge binds basic amino acid - cationic chromatography
Different proteins bind with different affinity
Eluted with increasing
amount of salt (NaCl or KCl)
Different proteins elute at
different salt concentrations
Size Exclusion Gel-filtration
Affinity Chromatography
• Uses specific binding properties of molecules/proteins
• Stationary phase has a polymer that can be covalently linked to
a compound called a ligand that specifically binds to protein
Electrophoresis
• charged particles migrate in electric field toward opposite
charge
• Proteins have different mobility:
• Blue dye is negative
• Everything runs according
to size!!
• Agarose used as matrix for nucleic acids
• Polyacrylamide used mostly for proteins
2-D Gel Electrophoresis
IEF (Isoelectric Focusing)
• Powerful technology to separate
proteins.
• Separates thousands of proteins
onto polymer gel.
• Allows physical properties of
proteins to be picked out
separately for analysis and
identification.
• First dimension is IEF to
separate by pI by pH
DiGE
Difference gel electrophoresis
Jeremy Keirsey – in house expert
[email protected]
DIGE – a visual 2D approach
Clinical drug stimulated cell line
Use Mass Spectrometry for protein
identification, charge state, structure
information, etc
Technical Issues
Where do you think your protein is?
“It’s exported to the plasma” or “it’s taken up by neuro-transmitters” or “it’s in
the blood”. Examples:
“Exported to plasma” – proteins go through the cell wall by
themselves, or with carrier associations. The researcher looks for
these by examining the cell in growth media and harvesting
media.
strategy: precipitate protein out of growth media (AmSO4
only, as TCA or Chloroform as the latter may cause their
protein to inactivate). Be careful! Media has a LOT of
manufacture added proteins that can interfere!
Examples Continued…
“It’s a neurotransmitter” – very lipidy samples. May also be
extremely glycosylated. Part of the protein may be hydrophobic and
part hydrophilic!
strategy: you will need to do some prep so that the
lipids and/or sugars will not interfere with downstream experiments
“It’s in the blood” – Ok! One of the most common proteins in blood
is hemoglobin. It is so abundant that it may interfere as well as the
iron!
strategy: remove hemoglobin by affinity
chromatography and albumen by CIBA-Blue
chromatography
What are you looking for?
“A signal protein” or “A nuclear protein” or “A structural protein”…answers
give you a great place to start! Examples:
Signal Proteins are post-translationally modified, in low
abundance and most copies of the protein will NOT have the
signal on it.
strategy: look for phosphorylations or nitrations on
2D, or use ProQ or nitro-tyrosine antibodies on 2D or
western blot
Nuclear Proteins locate to the nucleus. A large percentage of
them are positively charged and can be complexed with the
nuclear membrane
strategy: enrich for the nucleus only. Use cationic
chromatography to separate out.
Protein Interactions
When analyzing a new protein, ask – to what proteins
does it bind?
◦ strategy: Use new protein as an affinity agent to
isolate its binding partners
◦ Bind protein or TAG-protein to resin and run fresh lysate over it
◦ May detect low affinity, transient, or cellular environment
specific interactions – protein can be crosslinked to its ligand!
◦ Maybe use an immunoprecipitation or CO-IP
Enrich for Modifications!
Glycosylation?
Total = concavalin (ConA) Wheat Germ
Agglutinin(WGA)
Mannose = Concavalin, snowdrop lectin,
lentil lectin
Sialic Acid = Wheat Germ agglutinin,
Elderberry lectin
O-glycan = Jacalin, Peanut Agglutinin
Enrich for Phosphate
IMAC (immobilized metal) and strong
cation exchange
Enrich for Nitration
Antibody IP or CO-IP
Enrich for Acetylation
Antibody IP for acetyl-lysine
Not seeing what you want?
- Are the proteins expressing in the cell lines he is looking in?
- Were all the cells grown all at the same time? Harvest at
EXACTLY the same time?
- The cell has its own growth phases…the proteome will change THE ENTIRE
TIME!
- Many larger protein-protein interactions will bind to the inner
side of the membrane walls or to structural proteins
- Make sure as they check their soluble fraction – they ALSO check their
pellet for their proteins! Many just think “cell debris” and throw them
away!
- Is the pH right for the protein in question?
If the buffer is ANYWHERE near the pI of the protein, the protein will
precipitate out of the solution.
IS IT A TECHNICAL PROBLEM?
…Affects
proteome
and
metabolome!
Structural
Proteins
AND
glycolytic
pathways will
all
upregulate!
WASH WITH MEDIA, NOT PBS.
EXCHANGE MORE OFTEN WITH ½ NEW MEDIA!
Pathways
affected:
Glucose
Inositol
Betaine
Taurine
Choline
Na+
K+
Proline
JNK-p38
‘ERK-typeMAP kinases’
‘Tyrosine
Kinases’
+ HUNDREDS MORE!
Distribution among functional categories of the 500 most abundantly expressed proteins.
Burkhart J M et al. Circulation Research. 2014;114:12041219
Copyright © American Heart Association, Inc. All rights reserved.
ARE YOU LOOKING FOR A NEEDLE IN A HAYSTACK?
We can help you!
At OSU, we examine issues that scientists deal with
concerning dependable and reproducible data from
biological experiments!
- isolate proteins - Study them alone or Study them in combination
- Cutting edge science requires delicate and complicated
customized approaches
- Isolate proteins
- Assure that biological activity is maintained, if desired
We give advice on most experimental design to enhance the probability of
success!
Protein Services at OSU Proteomics
Just ask! •
Protein Growth, Induction and Expression, Protein purification
•
•
Subcloning into recombinant cell lines, Plasmid design
DIGE
•
•
Develop novel protein protocols, individualized for experiment
Selective subfractionation, Salt fractionation, Enrichment, Solubility screening,
Inclusion body isolation
Western Blotting, Far Western Blotting, Immunoprecipitation and Co-
•
immunoprecipitation, Protein-Protein interaction studies
•
Classic chromatography:
Affinity –Tag purification, ionic exchange, HIC reverse phase, SEC gel
chromatography 100,300, Immobilized metal affinity chromatography (IMAC),
Heparin affinity: Protein A/G affinity column, ENDOTOXIN removal
•
SDS-PAGE and DNA Electrophoresis, reduced and/or non-reduced
•
•
•
ProQ, LavaPurple, Sypro and other gel staining
Fluorescent and Bradford Protein Quantitation
Mass Spectrometry for protein identification
THANKS FOR LISTENING!
You can find us at…
Mass Spec and Proteomics and
Protein Expression and Purification
Facility
Biomedical Research Tower Room 250
460 West 12th Street
Columbus, Ohio
Lab: 614-247-8789
Arpad Somogyi, PhD – [email protected]
Cindy L. James, PhD – [email protected]