sample prep cindy - ohio state university prep_i_cindy.pdf · things to ask before sample prep and...
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Proteomics
GET THE BIOLOGICAL ANSWERS YOU WANTSAMPLE PREP AND SEPARATIONS
Cindy L. James, PhDProtein Biochemist
Our life is maintained by molecular network systems
(From ExPASy Biochemical Pathways; http://www.expasy.org/cgi-bin/show_thumbnails.pl?2)
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Protein interactions are at the core of the entire system of any living cell……AN ABSOLUTE REQUIREMENT TO UNDERSTAND!
Same genome, different proteome
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21,000 genes to encode a half million proteins!
DNA
RNA
PROTEINS
IE: Alternative splicing ‐ In humans, many genes contain multiple introns and make multiple proteins!!
3 4 51 2
1 2 3 54intron 2 intron 3 intron 4intron 1
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!
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Hydrophobic, lipid sticks to it, will show in lysis pellet
Hydrophillic, can shear and show in supernatant
Binds and shows with RNA, Reflects higher molecular weight,Very Positively charged
Proteins come in many sizes, shapes and chemistries
Phospholipid bylayer
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!!
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Not Every Sample or Cell‐line has the same proteome!
Our samples come from:
Bacteria FecesFood HairPlants RootsSediment SeedsSludge TreeTissue UrineNasal secretion YeastBlood PlasmaSalivaUrineBiopsy
For Example: Tissue samples obtained from a biopsy, or 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
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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 solvents may degrade certain compounds
• Its good to have some idea of what metabolites you want to extract
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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?
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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 and IGEPAL CA-630).
IP FOR PROTEIN-PROTEIN STUDIES:- Use IGEPAL CA-630, it is less denaturing and better for kinase activity and protein interactions
Do not use RIPA – it can disrupt protein:protein interactions. IGEPAL CA-630 is a non-ionic commonly used detergent in cell lysis buffers for immunoprecipitation and western blot.
Are you looking for phosphorylation of proteins, protein-protein interactions, or membrane boundIGEPAL 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 proteinTry RIPA. RIPA buffer can give lower background in immunoprecipitation, but can denature some proteins.
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Protein location Buffer recommendedWhole Cell IGEPAL CA-630 or RIPACytoplasmic (soluble) Tris-HClCytoplasmic (cytoskeletal bound) Tris-TritonMembrane bound CJ’s MemLysis or RIPANuclear IGEPAL CA-630 or use nuclear fraction protocolMitochondria IGEPAL CA-630 or use mitochondrial fraction protocol
Enrich for your elusive target protein by fractionation. Can load more of the protein per gel lane. Will help removal of potentially cross-reactive proteins from unused fractions.
FOR MEMBRANE PROTEINSCJ’S MemLysis Buffer – used for signal and membrane proteinsA chaotrope plus aminosulfobetaine OR similar to compete with lipid/hydrophobic amino acid that involve imbedded membrane proteins.
Detergent fatty-acid chain should mimic the lipids it wants to dissolve.Since cell walls are different for membrane proteins, the detergent used should have similar number of carbons and polar-head ionics to match it!
Separation by subfraction
GET YOUR PROTEINS AWAY FROM ALL THE OTHER KIDS IN THE PLAYGROUND!
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Differential Centrifugation
Zonal centrifugation
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Common Separation techniques
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
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Know the charge of your protein!
Know the pI of your proteins of interest!
INDUSTRY RULE:
If your buffer is at the pI of the protein(s) you are after, you WILL lose your protein in precipitate!
Every protein has a pI (point where there is an OVERALL ZERO charge, not where there are no charges on your protein!)
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Isoelectric Points of Several Common Proteins
Protein pIPepsin 1.0Ovalbumin (hen) 4.6Serum albumin (human) 4.9Tropomyosin 5.1Insulin (bovine) 5.4Fibrinogen (human) 5.8g-Globulin (human) 6.6Collagen 6.6Myoglobin (horse) 7.0Hemoglobin (human) 7.1Ribonuclease A (bovine) 9.4Cytochrome c (horse) 10.6Histone (bovine) 10.8Lysozyme (hen) 11.0Salmine (salmon) 12.1
Using pI to separate your protein(s)
[H+]pI ~5
Protein becomes increasingly -chgProtein becomes increasingly +chg
[OH-]
pI ~8.5
What buffer would you use? Which pH range is best?
PROTEIN A
PROTEIN B
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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.
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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.
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Separating by Column Chromatography
Column Chromatography
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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 atdifferent salt concentrations
Size Exclusion Gel-filtration
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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
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• 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
2‐D Gel ElectrophoresisIEF (Isoelectric Focusing)
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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!
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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 down-stream 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.
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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!Total = concavalin (ConA) Wheat Germ
Glycosylation? Agglutinin(WGA)Mannose = Concavalin, snowdrop lectin, lentil lectinSialic Acid = Wheat Germ agglutinin, Elderberry lectinO-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
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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!
Pathways affected:GlucoseInositolBetaineTaurineCholineNa+K+ ProlineJNK-p38‘ERK-type-MAP kinases’‘Tyrosine Kinases’
+ HUNDREDS MORE!
StructuralProteins AND glycolytic pathways will all upregulate!
WASH WITH MEDIA, NOT PBS. EXCHANGE MORE OFTEN WITH ½ NEW MEDIA!
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Distribution among functional categories of the 500 most abundantly expressed proteins.
Burkhart J M et al. Circulation Research. 2014;114:1204-1219
Copyright © American Heart Association, Inc. All rights reserved.
We can help you!ARE YOU LOOKING FOR A NEEDLE IN A HAYSTACK?
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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 and 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!
delicate complicated
SERVICES at OSU Proteomics• 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
Just ask!
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Mass Spec and Proteomics andProtein Expression and Purification
Facility
Biomedical Research Tower Room 250460 West 12th StreetColumbus, OhioLab: 614-247-8789
Arpad Somogyi, PhD – [email protected] L. James, PhD – [email protected]
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