Lecture 8
Chromatography in proteomics
A.
B.
C.
Affinity
Ion Exchange
Reversed-phase
Oct 2010 SDMBT
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Affinity Chromatography
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 binding
an 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.
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Affinity
Examples of biological interactions used in affinity chromatography
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Affinity
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 protein
The immobilized ligand / support matrix combination should be
highly selective stationary phase
matrix
• 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.
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Affinity
Properties of ligands
• Ligand should have chemical properties that allow easy
immobilization to a matrix.
• 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.
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Affinity
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.
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Affinity
• 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.
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Affinity
• 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.
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Affinity
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.
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Affinity
• 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
6 carbon chain – hydrophobic may attract hydrophobic proteins
Stationary phase packing material – usually hydrophilic
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Affinity
Major affinity systems with pre-immobilized ligands
Protein 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.).
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Table 4-4.
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Affinity
• 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.
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Affinity
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.
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Affinity
Elution Method
Because ligand-ligand interaction complex
mixture of hydrophobic, ionic forces – elution
mechanisms may be complex
• Elution can be done with any agent that disrupts the ligandligand 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.
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Imidazole competes with protein
to bind to the IMAC sepharose 15
Affinity - purification of protein complexes
Tandem Afffinity Purification (TAP)
A method to find out protein-protein interactions
TAP tag consists of (i) calmodulin-binding peptide (ii) TEV protease cleavage site
(iii) Protein A
-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)
-All other proteins are washed away
-TEV protease used to cut off protein A
in TAP tag
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Affinity - purification of protein complexes
Tandem Afffinity Purification (TAP)
-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 calmodulin
binding peptide
-Protein-protein interactions in complex are not disrupted so far.
-Break up the complex by SDS and run SDS PAGE or trypsin digestion
From: Is proteomics heading in the wrong direction?
Lukas A. Huber, Nature Reviews Molecular Cell Biology 4, 74-80 (January 2003)
Oct 2010 SDMBT
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 anhydrous
methanol and thionyl chloride (SOCl2)
O
O
HO
R
O
R
O
R
H3C
O
R
Acidic amino acids will also complex to Fe3+ in IMAC
So 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
• It separates proteins based on differences in their accessible
surface charges;
• Charged proteins and other ions compete to bind to
the oppositely charged groups on an ion exchanger
CATION-EXCHANGER attracts cations
ANION-EXCHANGER attracts anions
Anion-exchange
resin
Positive charge
Attracts negative ions
(eg anionic proteins)
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Protein
accessible charge
negative (-) 19
anionic
IEX
Cation-exchange resin
Negative charge
attracts positive ions
(eg cationic proteins)
Charged functional
Group covalently bonded
To resin/stationary phase bead
Protein
accessible charge
positive (+)
cationic
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IEX
• 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 Strength
i.e. concentration Na+ and Cl-
Attraction between
the protein and the ion-exchanger
Na+ competes with the
Cationic protein to bind to the
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ion-exchanger
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IEX
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
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IEX
• 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.
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IEX
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.
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IEX
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.
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IEX
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IEX
The stationary phase: ion exchangers
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)
Functional Groups
- Functional groups are bonded covalently/
permamently to the packing material.
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IEX
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)
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IEX
• 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..
SCX
Strong Cation
Exchanger
strong acid functional group
(complete ionization)- eg sulphonic
acid group is covalently bonded to
resin particle (S)
SAX
Strong Anion
Exchanger
completely ionized salt functional
group eg quartenary amine group is
covalently bonded to resin particle
(Q)
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IEX
• 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
WCX
Weak Cation
Exchanger
weak acid functional group
(incomplete ionization)- eg
carboxymethyl acid group is
covalently bonded to resin particle
(CM)
WAX
Weak Anion
Exchanger
weak base functional group eg
diehylaminoethyl group is
covalently bonded to resin particle
(DEAE)
* 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.
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IEX
• 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.
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IEX
Table 3-1. Functional groups used in ion exchangers
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IEX
The mobile phase: buffers and salts
Buffer 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+.
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IEX
• 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.
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IEX
Experimental planning and preparation
Choosing 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, protein
Neutral (uncharged) will not bind to IEX resin
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IEX
(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.
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IEX
Examples
Many enzymes and blood
proteins
pI
acidic
Limitations
Type of IEX
Stable only at neutral
pH
Anion exchange
since protein will be
anions at neutral pH
pH 7
Many regulatory proteins
(eg cytokines and growth
factors)
basic
Only soluble in acidic
media
pH < 7
Cation exchange
Since proteins will
be cations at acidic
pH
Many other biomolecules have a solubility and stability pH range that
encompasses their pI, so that either anion or cation exchange can
be used.
(ie pI>7)
(ie pI < 7)
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IEX
• 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 counterion
Eg cation exchanger – H+, Na+, K+
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IEX
-- 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;
If media have no charge – then cannot attract ions
- cannot be ion exchange
-- In addition, weak ion exchange media can take much longer
to equilibrate because the column itself has a significant
buffering capacity.
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Reversed-phase RP
-Reversed-phase chromatography – stationary phase made of
porous silica beads modified by long hydrophobic alkyl C18H37 (C18) chains
- Stationary phase is hydrophobic and attracts
hydrophobic molecules
-Many different silica base materials available
e.g. Zorbax, Poroshell etc – different pore
sizes and particle sizes available
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Reversed-phase RP
-Typical mobile phases are water/methanol
or 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
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
-SH more polar than disulfide linked peptides
Tryptic digest was separated on a
ZORBAX 300SB-C18 column before
(bottom panel) and after (top panel)
reduction with TCEP - Tris(2carboxyethyl)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 Proteins
Hsin‐Yao Tang, David W. Speicher, Current Protocols in Protein Science, 2004
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From Determination of Disulfide‐Bond Linkages in Proteins
Hsin‐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 Spectrometry
Andrew J. Link, Jennifer L. Jennings, Michael P. Washburn, Current Protocols in Protein Science 2004
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MudPIT approach to proteomics
Cell 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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MudPIT approach to proteomics
All compounds eluting from the RP-HPLC ionised straightaway by
electrospray 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
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MudPIT vs 2-D Gels
2D-PAGE & MALDITOF
Peptide HPLC-MS-MS
What is being separated?
Proteins
Peptides (after tryptic
digestion)
How are they separated?
pI (charge) (IEF) then
Size (SDS-PAGE)
Charge (SCX) then
Hydrophobicity (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 MSMS Spectrum
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MudPIT vs 2-D Gels
2D-PAGE & MALDITOF
Peptide HPLC-MSMS
Advantages
Cheap
Reproducible and can
be automated
Good for highLower detection limits
abundance proteins.
Can detect posttranslational
modification of protein
Disadvantages
Poorer reproducibility,
labour intensive
High pI proteins and
hydrophobic proteins
not captured
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