Proteomics

GET THE BIOLOGICAL ANSWERS YOU WANTSAMPLE PREP AND SEPARATIONS

Cindy L. James, PhD Protein Biochemist

Our life is maintained by Our life is maintained by molecular network systemsmolecular 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!

GLYCOLYTIC PATHWAY

Same genome, different proteome

Only 21,000 genes to encode a half million proteins!Only 21,000 genes to encode a half million proteins!

DNADNA

RNARNA

PROTEINSPROTEINS

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!

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 What do you want to know?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!!GOOD SAMPLE PREPARATION IS ESSENTIAL!!

Not Every Sample or Cell-Not Every Sample or Cell-line has the same line has the same

proteome! 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 Not all Proteins are produced by the cell the cell

in equal amounts!in equal amounts!

Favored research drug targets (signal proteins) are actually lowlow 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 Some Experiment ChallengesChallenges

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 existsNo universal extraction method exists

• Some solvents may degrade certain compounds

• Do you know the characteristics of metabolites you want to extract?

Notes on Sample Notes on Sample PreparationPreparation

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 Things to ask before sample prep and whyprep 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 TISSUESOPEN YOUR CELLS AND TISSUES

Does your lysis buffer make a difference? Does your lysis buffer make a difference? YES!!!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 proteinTry RIPA. RIPA buffer can give lower background in immunoprecipitation, but can denature some proteins.

Detergents do not always denature protein, is more like

ASB-14

CHAPS

SDS

IGEPAL-CA360

Polar head groups

Phobic tails

PHOSPHOLIPIDS

Protein location Buffer recommendedWhole Cell RIPACytoplasmic (soluble) Tris-HClCytoplasmic (cytoskeletal bound) Tris-TritonMembrane bound CJ’s MemLysis or RIPANuclear use nuclear fraction protocol and RIPAMitochondria use mitochondrial fraction protocol, high salt & IGEPAL

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 proteins. A 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 subfractionSeparation by subfraction

GET YOUR PROTEINS AWAY FROM ALL THE OTHER KIDS IN THE PLAYGROUND!

Differential Centrifugation

Zonal centrifugation

Common Separation techniquesCommon 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

Know the charge of your protein!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!)

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

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 ChromatographySeparating 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 atdifferent 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

• 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 Electrophoresis2-D Gel ElectrophoresisIEF (Isoelectric Focusing)IEF (Isoelectric Focusing)

DiGE Difference gel electrophoresisDifference gel electrophoresis

Jeremy Keirsey – in house expertKeirsey.1@osu.edu

DIGE – a visual 2D approach

Clinical drug stimulated cell line

Technical IssuesTechnical Issues

Where do you think your Where do you think your protein is?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” – p– 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 Examples Continued…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 What are you looking for?for?

“A signal protein” or “A nuclear protein” or “A structural protein”…answers give you a great place to start! Examples:

Signal Proteins 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 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 InteractionsProtein 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

Not seeing what you Not seeing what you want? 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 IS IT A TECHNICAL PROBLEM?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!

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!We can help you!ARE YOU LOOKING FOR A NEEDLE IN A HAYSTACK?ARE YOU LOOKING FOR A NEEDLE IN A HAYSTACK?

At OSU, we examine issues that At OSU, we examine issues that scientists deal with concerning scientists deal with concerning dependable and reproducible data dependable and reproducible data from biological experiments!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!

SERVICES at OSU SERVICES at OSU ProteomicsProteomics

• 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!Just ask!

Mass Spec and Proteomics andMass Spec and Proteomics and Protein Expression and Purification Protein Expression and Purification FacilityFacility

Biomedical Research Tower Room 250460 West 12th StreetColumbus, OhioLab: 614-247-8789

Arpad Somogyi, PhD – somogyi.16@osu.eduCindy L. James, PhD – james.456@osu.edu

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