Lecture 8 Chromatography in proteomics Affinity Ion Exchange Reversed-phase

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Oct 2010 SDMBT Lecture 8 Chromatography in proteomics A.Affinity B.Ion Exchange C.Reversed-phase 1

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Lecture 8 Chromatography in proteomics Affinity Ion Exchange Reversed-phase. Affinity Chromatography. Principles of Affinity Chromatography. Affinity chromatography is based on biospecific binding interactions between a ligand chemically bound (immobilised) to the - PowerPoint PPT Presentation

Transcript of Lecture 8 Chromatography in proteomics Affinity Ion Exchange Reversed-phase

Page 1: Lecture  8 Chromatography in proteomics Affinity Ion Exchange Reversed-phase

Oct 2010 SDMBT

Lecture 8Chromatography in proteomicsA.AffinityB.Ion ExchangeC.Reversed-phase

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Principles of Affinity Chromatography• Affinity chromatography is based on biospecific binding interactions between a ligand chemically bound (immobilised) to the chromatographic packing and a target molecule in the sample.

Some examples of biospecific bindingan antigen to an antibody;

a substrate, inhibitor or cofactor to an enzyme; a regulatory protein to a cell surface receptor; etc.

• The forces involved in the binding can be ionic or hydrophobic interaction.

• In affinity chromatography, one member of the ligand pair is immobilized (i.e. covalently bonded/coupled) as a bonded phase.

Affinity Chromatography

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Examples of biological interactions used in affinity chromatography

Affinity

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a spacer arm or linker -to place some distance between the bound ligand and the support matrix to improve protein accessibility to the ligand

Immobilized ligand should only bind to one specific proteinThe immobilized ligand / support matrix combination should be highly selective stationary phase

• The support matrix (packing or base material) and spacer arm (linker) themselves should have minimal binding interaction (nonspecific adsorption) with any of the molecules in solution.

matrix

Affinity

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Properties of ligands

• The ligand must be able to form reversible complexes with the protein to be isolated or separated.

 • The complex formation equilibrium constant should be high enough for the formation of stable complexes or to give sufficient retardation.

• It should be easy to dissociate the complex by a simple change in the medium, without irreversibly affecting the protein to be isolated or the ligand.

• Ligand should have chemical properties that allow easy immobilization to a matrix.

Affinity

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Types of ligands

• Monospecific low molecular weight ligands -- these ligands bind to a single or a very small number of proteins.

• Group-specific low molecular weight ligands -- the largest group of ligands, eg a wide variety of enzyme cofactors, biomimetic dyes, boronic acid derivatives, and a number of amino acids and vitamins.

The target proteins are most often enzymes and the most thoroughly studied are the NAD+- and NADP+-dependent dehydrogenases and kinases.

Affinity

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• Monospecific macromolecular ligands It is through specific protein-protein interactions, e.g. the binding of fibronectin to gelatin; antithrombin to thrombin and heparin; transferrin receptor to transferrin; antibody to antigen; etc.

• Immunoadsorbents It is through high specificity of antibodies. Both antigens and antibodies can be used as affinity ligands.

The traditional immunoadsorbents are based on polyclonal antibodies. The modern immunoadsorbents are based on monoclonal antibodies.

Affinity

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• Group-specific macromolecular ligands

This group includes several ligands that have found widespread popularity;

e.g., lectins such as concanavalin (Con A) and lentil for glycoproteins; protein A and protein G for antibody; calmodulin for calcium-dependent enzymes; etc.

Affinity

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Choice of spacer arm (linker)

• Low molecular weight ligands might show poor function due to low steric availability of the ligands.

• The use of a spacer arm or linker can solve this problem.

• Commonly used linkers are aliphatic, linear hydrocarbon chains with two functional groups located at each end of the chain.

• One of the groups (often a primary amine, -NH2) is attached to the matrix, whereas the group at the other end (usually a carboxyl, -COOH, or amino group, -NH2) is attached to the ligand.

Affinity

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• The most common spacers are 6-aminohexanoic acid [H2N- (CH2)6-COOH], hexamethylene diamine [H2N- (CH2)6-NH2], and l,7-diamino-4-azaheptane (3,3-diaminodipropylamine).  

• A drawback with the hydrocarbon linkers, especially the longer ones, is that they can give rise to unwanted nonspecific interactions – hydrophobic interactions

Stationary phase packing material – usually hydrophilic

6 carbon chain – hydrophobic may attract hydrophobic proteins

Affinity

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Major affinity systems with pre-immobilized ligandsProtein A / G affinity chromatography

• Protein A / G, which is a specific protein originally extracted from the surface of certain gram positive bacteria i.e. staphylococcal and streptococcal, but now usually made recombinantly, is immobilized onto eg Sepharose beads

• These proteins selectively bind to a broad range of antibody molecules, thus forming an affinity column for antibodies.

• Proteins A and G differ in both their species and subclass specificity for antibody binding (shown in Table 4-5.).

Affinity

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Table 4-4.

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• When both proteins will work, protein A is always recommended because of the lower cost and harsher conditions which can be used in cleaning and regeneration.

• The most important type of antibody bound by protein G and not normally by protein A is mouse IgG1 which is the most common subclass of monoclonal antibodies. However, the addition of high salt (2-3 M NaCl) with high pH (8-9) to the binding / wash buffer will cause these antibodies to bind.

Affinity

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Immobilized metal affinity chromatography (IMAC)

• A metal chelating group (typically imidodiacetate) is immobilized;

• a multivalent transition metal ion (typically Cu2+>Ni2+>Zn2+ Co2+

or Fe2+ in order of binding strength) is bound in such a way that one or more coordination sites are available for interaction with proteins.

• Certain surface amino acids (primarily histidines) bind specifically to these free coordination sites.

• The separation is based on the surface concentration of these amino acids.

Affinity

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Elution Method

• Elution can be done with any agent that disrupts the ligand- ligand interaction.

• The most common technique is to employ a shift to acidic pH (usually to pH 2-4), which is used extensively for protein A/G and for antibody ligand affinity methods.

• Affinity elution is usually in the form of a step gradient.

• For IMAC, a gradient elution in imidazole concentration is normally used. Imidazole is the active functionality in histidine which binds to the metal coordination site.

Because ligand-ligand interaction complexmixture of hydrophobic, ionic forces – elutionmechanisms may be complex

Imidazole competes with proteinto bind to the IMAC sepharose

Affinity

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Affinity - purification of protein complexesTandem Afffinity Purification (TAP)

A method to find out protein-protein interactions

-DNA coding the TAP tag is inserted after the DNA for the protein of interest-Organism produces a recombinant protein with the TAP tag-The protein of interest is free to associate with other proteins-Cell is lysed and protein complex with TAP tag is released to bind to IgG sepharose beads (IgG+protein A have specific attraction)

TAP tag consists of (i) calmodulin-binding peptide (ii) TEV protease cleavage site (iii) Protein A

-All other proteins are washed away-TEV protease used to cut off protein Ain TAP tag

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Affinity - purification of protein complexesTandem Afffinity Purification (TAP)

From: Is proteomics heading in the wrong direction?Lukas A. Huber, Nature Reviews Molecular Cell Biology 4, 74-80 (January 2003)

-Residual protein binds to calmodulin beads through calmodulin binding peptide-Elute the protein with a buffer containing EGTA-EGTA chelates Ca2+ which is responsible for binding calmodulin to the calmodulinbinding peptide-Protein-protein interactions in complex are not disrupted so far.-Break up the complex by SDS and run SDS PAGE or trypsin digestion

http://www.bio.davidson.edu/Courses/Molbio/calmod/calmodulin.html

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Affinity - purification of phosphopeptides by IMAC

-Peptide mixture is methyl esterified with anhydrousmethanol and thionyl chloride (SOCl2)

ROH

RO

O

RO

RO

O

CH3

Acidic amino acids will also complex to Fe3+ in IMACSo esterification ensures only phosphorylated amino acids trapped Fe3+

-Methyl esterified peptide passed through Fe3+IMAC column-Traps only phosphorylated peptides -Phosphorylated peptides eluted with phosphate buffer – phosphate competes to bind to Fe3+)

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Ion-exchange chromatography (IEX)Fundamental Concepts

• Charged proteins and other ions compete to bind to the oppositely charged groups on an ion exchanger

• It separates proteins based on differences in their accessible surface charges;

Anion-exchangeresin Positive chargeAttracts negative ions (eg anionic proteins)

Proteinaccessible chargenegative (-)anionic

CATION-EXCHANGER attracts cationsANION-EXCHANGER attracts anions

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IEX

Proteinaccessible chargepositive (+)cationic

Cation-exchange resin Negative chargeattracts positive ions (eg cationic proteins)

Proteinaccessible chargepositive (+)cationic

Charged functionalGroup covalently bondedTo resin/stationary phase bead

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• The interaction between small molecules and an ion exchanger depends on the net charge and the ionic strength of the medium – ionic strength,

• When the concentration of competing ions is low, the ions of interest bind to the ion exchanger, whereas when it is high, they are desorbed;

Ionic Strengthi.e. concentration Na+ and Cl-Attraction betweenthe protein and the ion-exchanger

Na+ competes with theCationic protein to bind to theion-exchanger

IEX

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Principle of ion-exchange chromatography. Species with several positive charges (A3+) are adsorbed to the column; those with few charges move slowly, whereas those with zero net charge or a net negative charge pass through the column unretained ie they are not well separated

IEX

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• The interaction between a protein and an ion exchanger depends on -- the net charge; -- the ionic strength of the mobile phase; -- the surface charge distribution of the protein; -- pH; -- the nature of particular ions in the solvent; -- additives e.g. organic solvents -- properties of the ion exchanger.

• The more highly charged a protein is, the more strongly it will bind to a given, oppositely charged ion exchanger.

• More highly charged ion exchangers, usually bind proteins more effectively than weakly charged exchangers.

IEX

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The pH parameter

• pH determines charge on both the protein and the ion exchanger, therefore it is one of the most important parameters in determining protein binding.

• At pH values far away from the pI, proteins bind strongly and in practice do not desorb at low ionic strength.

• Near to its pI, the net charge of a protein is less and consequently it binds less strongly.

• An ion exchanger is normally used in conditions where its charges will not be significantly changed (titrated) by small shifts in pH.

IEX

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Influence of Ions

• The proteins compete with other ions in the mobile phase buffer/solvent to bind to the charged groups on the ion exchanger;

• If concentration of competing ions low, proteins preferably bound through interactions between several charged groups on the proteins and oppositely charged groups on the ion exchanger.

• If concentrations of competing ions high, the proteins will start to be displaced from the ion exchanger. The most weakly bound are displaced and eluted from the column first.

IEX

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IEX

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- Functional groups are bonded covalently/permamently to the packing material.

The stationary phase: ion exchangers

Functional Groups

The stationary phase in ion-exchange (usually known as an ion-exchange resin) is made of 2 components- packing or base material (section 3.3.2)- functional group or bonded phase (see below)

IEX

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eg Cation Exchange resin – the bound functional group has negative charge- the counterion associated with the functional group is positive (cation)- the counterion can be easily displaced by other cations since not permamently bound to resin particle – exchange cations

Summary:•, Cation exchange column separates cations (positive charge)• Anion exchange column separates anions (negative charge)

IEX

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• Anion or cation exchange functional groups can be classified as either “weak” or “strong”;

• Strong ion exchange resins - charge of resin is independent of the pH of the mobile phase..

SCXStrong Cation Exchanger

strong acid functional group (complete ionization)- eg sulphonic acid group is covalently bonded to resin particle (S)

SAXStrong Anion Exchanger

completely ionized salt functional group eg quartenary amine group is covalently bonded to resin particle (Q)

IEX

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• Weak ion exchange groups - may gain or lose electrical charge as the pH of the mobile phase changes. – can be used as over limited pH range

* Note: the terms “strong” or “weak” do not refer to the strength of the binding but only to the effect of pH on the charge of the functional groups.

WCXWeak Cation Exchanger

weak acid functional group (incomplete ionization)- eg carboxymethyl acid group is covalently bonded to resin particle (CM)

WAXWeak Anion Exchanger

weak base functional group eg diehylaminoethyl group is covalently bonded to resin particle (DEAE)

IEX

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• All cation exchangers have a limiting pH below which they cannot be used. As a rule of thumb, the pKa is suggested as the lower limit.

• Weak anion exchangers have an upper pH limit for their use. For the quaternary amines, there is no upper limit as they will not lose the charge whatever the pH.

IEX

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Table 3-1. Functional groups used in ion exchangers

IEX

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The mobile phase: buffers and saltsBuffer pH and concentration• Normally the concentration of buffer salts during protein adsorption is low, around 0.01 to 0.05 M.• Criteria for choosing buffer: (i) The buffer should have a high capacity, preferably with the pKa of the buffering species less than 0.5 units from the working pH;

(ii) The buffering species should not interact with the ion exchanger.

• For an anion exchanger, a positive buffering ion, such as Tris (pKa 8.2), is often used and usually with Cl- as the counterion.

• For a cation exchanger, a negatively charged buffering ion is recommended, e.g., phosphate, acetate, and the counterions are mostly Na+ and K+.

IEX

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• A nonbuffering salt, such as NaCl, is usually added to the buffer to elute proteins from an ion exchanger

• Elution methods may also include changes in pH along with (or instead of) ionic strength increases.

Because the pH can affect the charge of the sample molecules as well as the charge of the bonded phase (i.e. weak ion exchange media). Changes in pH can thus be used to weaken or eliminate charge-charge interactions, thereby causing elution.

IEX

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Experimental planning and preparationChoosing an ion exchange column

• Use anion or cation exchange column.

(i) It depends on the charge characteristics and the effect of pH on stability and solubility of both the target molecule itself and the other molecules in the sample.

(ii) To maximize binding strength, select an operating pH range that is either above or below the pI of the target, based on where the biomolecule is most stable and soluble.

If pH=pI of protein, proteinNeutral (uncharged) will not bind to IEX resin

IEX

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(iii) The ion exchanger is then chosen by the following rule:

-- If pH of medium > pI, the protein molecule is negative. Try anion exchange column first.

-- If pH of medium < pI the protein molecule is positive. Try cation exchange column first.

IEX

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Many other biomolecules have a solubility and stability pH range thatencompasses their pI, so that either anion or cation exchange canbe used.

Examples pI Limitations Type of IEX

Many enzymes and blood proteins

acidic Stable only at neutral pH

Anion exchangesince protein will be anions at neutral pH

Many regulatory proteins (eg cytokines and growth factors)

basic Only soluble in acidic media

Cation exchangeSince proteins will be cations at acidic pH

(ie pI < 7)

pH 7

(ie pI>7)

pH < 7

IEX

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• Use a weak or strong ion exchange functional group.

-- For most biomolecules and pH ranges, either strong or weak ion exchange media may be used;

-- As a starting point for method development, use strong ion exchange media, since they operate over a broader pH range and equilibrate more easily than weak ion exchange media.

Need to soak weak ion exchange media with buffer containing a counterionEg cation exchanger – H+, Na+, K+

IEX

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-- In extreme pH conditions (pH>10 for anion exchange and

pH<3-5 for cation exchange), weak ion exchange media lose

most of their charge, and thus bind molecules very weakly

or not at all;

-- In addition, weak ion exchange media can take much longer

to equilibrate because the column itself has a significant

buffering capacity.

If media have no charge – then cannot attract ions- cannot be ion exchange

IEX

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Reversed-phase RP

-Reversed-phase chromatography – stationary phase made ofporous silica beads modified by long hydrophobic alkyl C18H37 (C18) chains

- Stationary phase is hydrophobic and attracts hydrophobic molecules

-Many different silica base materials availablee.g. Zorbax, Poroshell etc – different poresizes and particle sizes available

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-Typical mobile phases are water/methanolor water/acetonitrile mixtures

- Typically samples are eluted by a gradient of increasing non-polar solvent (methanol or acetonitrile) concentration. e.g. start with 2% increasing to 80% acetonitrile

- Usually buffered or acidified e.g. 0.1% trifluoroacetic acid, or formic acid (volatile acid) – if sample is for LC-MS

Reversed-phase RP

The more polar the compound (has more OH groups, C=O etc), shorter retention time.The less polar the compound (has more C-H, C-C bonds),longer retention time.

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Reversed-phase RP

Tryptic digest was separated on a ZORBAX 300SB-C18 column before (bottom panel) and after (top panel) reduction with TCEP - Tris(2-carboxyethyl)phosphine hydrochloride. Major peaks, which disappeared following reduction are indicated by T1 to T3. The peptide constituents of the complexes are shown in Figure 11.11.2. T2* and T3* indicate incomplete tryptic cleavages of T2 and T3, respectively. Single peptides that appear as a result of reduction are indicated in the top panel by their residue numbers.

From Determination of Disulfide‐Bond Linkages in ProteinsHsin‐Yao Tang, David W. Speicher, Current Protocols in Protein Science, 2004

-SH more polar than disulfide linked peptides

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From Determination of Disulfide‐Bond Linkages in ProteinsHsin‐Yao Tang, David W. Speicher, Current Protocols in Protein Science, 2004

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MudPIT approach to proteomics

Column is a SCX (strong cation exchanger) followed by a reverse phase column

Analysis of Protein Composition Using Multidimensional Chromatography and Mass SpectrometryAndrew J. Link, Jennifer L. Jennings, Michael P. Washburn, Current Protocols in Protein Science 2004

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MudPIT approach to proteomicsCell lysate

Digest with trypsin

Load samples on SCX with e.g. 5% acetonitrile 0.1% formic acid

Elute with increasing % acetonitrile – uncharged peptides separated by polarity

Change to higher ionic strength buffer e.g. add some 5% acetonitrile, 0.1 % formic acid 500 mM ammonium acetate to mobile phase to elute low charge peptides

Elute with increasing % acetonitrile – low charge peptides separated by polarity

Change to even higher ionic strength buffer to elute more highly charged peptides

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All compounds eluting from the RP-HPLC ionised straightaway byelectrospray ionisation

MS captures the molecular weight of peptides eluting at a particular time

Programme MS to select the top 4-5 peptides to fragment further

MS/MS spectra – can tell the amino sequence of part of peptide – identify protein

MudPIT approach to proteomics

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2D-PAGE & MALDI-TOF

Peptide HPLC-MS-MS

What is being separated? Proteins Peptides (after tryptic digestion)

How are they separated? pI (charge) (IEF) thenSize (SDS-PAGE)

Charge (SCX) thenHydrophobicity (RP)

How are they identified? Trypsin digest followed by MALDI-TOF

Directly into ESI-MS-MS

Basis of identification Peptide mass fingerprinting[Pattern of Mw of peptides]

Interpretation of fragmentation in the MS-MS Spectrum

MudPIT vs 2-D Gels

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2D-PAGE & MALDI-TOF

Peptide HPLC-MS-MS

Advantages CheapGood for high-abundance proteins.Can detect post-translational modification of protein

Reproducible and can be automatedLower detection limits

Disadvantages Poorer reproducibility, labour intensive

High pI proteins and hydrophobic proteins not captured

MudPIT vs 2-D Gels