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Abstract
Affinity chromatography represents a promising technique for decoding the proteomics universe.
While conventional affinity purification is being used in conjunction with two-dimensional
electrophoresis (2D-PAGE) and mass spectrometry (MS) for the study of proteomes and
subproteomes, scientists are still confronted with the need for specific and tailor-made affinity
ligands to target desired groups and families of proteins. Evidence has shown that, in many
situations, synthetic affinity ligands can circumvent inconveniences associated with the utilisation of
biological ligands for the chromatography-based purification of biomolecules. This review will
highlight the potential applications of affinity chromatography and synthetic de novo designed
ligands as separation tools for proteomics.
D 2005 Elsevier Inc. All rights reserved.
1. Introduction
* Corresponding author. Tel.: +44 1223 334157; fax: +44 1223 334162.
E-mail address: crl1@biotech.cam.ac.uk (C.R. Lowe).
0734-9750/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2005.05.001
JBA-05983; No of Pages 14
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2 A.C.A. Roque, C.R. Lowe / Biotechnology Advances xx (2005) xxx–xxx
unravel the secrets of the gene products, the proteins. The equivalence between genes
and the proteins they code for is generally a non-straightforward process, not only
because many post-transcriptional and post-translational modifications take place,
but many cellular regulation mechanisms also control the expression of genes.
Proteomics can be defined as the analysis of part or all of the protein complements of
a complex biological system at any given moment in time and it includes the
characterization and quantitation of protein expression, function and structure (Shi et
al., 2004). Proteome analysis requires techniques that could provide high throughput,
high sensitivity and resolution, as well as the possibility of quantifying and identifying
PTM (post-translational modifications) in proteins. Conventional experimental techni-
ques used in proteomics include two-dimensional electrophoresis (2D-PAGE) and mass
spectrometry (MS).
Two-dimensional electrophoresis is widely used for the separation and quantitation of
thousands of intact proteins in a single run. It also presents the possibility of applying
labelling techniques for the detection of PTM and comparative protein expression in
selected cell populations. It is therefore possible to use general protein detection
reagents for a holistic approach (organic dyes, silver stain, radiolabelling and fluorescent
stains, amongst others) and specific detection methods to reveal the status of proteins,
such as the level of phosphorylation (e.g. autoradiography 32P and 33P), glycosylation
(e.g. dansyl hydrazine), proteolytic modifications (e.g. zymographic assays for serine
proteases), S-nitrosylation (e.g. Biotin Switch method), arginine methylation (e.g.
immunoblotting) and ADP-ribosylation (e.g. 1,6-etheno NAD+) (Patton, 2002). Another
option employs recombinant-DNA strategies to couple affinity tags or reporter enzymes
to proteins and this approach has proven useful both at the detection and purification
levels. Examples of these affinity tags include the FLAG and oligohistidine peptides,
the green fluorescent protein and the enzyme h-glucuronidase. Differential display
proteomics is based on the comparison of different protein profiles — examples of this
approach using gel electrophoresis are difference gel-electrophoresis (DIGE) and
multiplexed proteomics (MP). Isotope-encoded affinity tagging is another powerful
strategy that combines affinity chromatography, gel electrophoresis and peptide mass
profiling by MS (Patton, 2002). Despite the developments achieved in 2D-PAGE, there
are still major technical disadvantages such as: poor reproducibility, difficult detection
of extremely acidic or basic proteins (pI b 3.5 and N9.5), hydrophobic or membrane-
located proteins, as well as proteins with extreme range of molecular weights, and
limitations on the amount of proteins that can be loaded and deficient detection of low
abundance proteins.
Mass spectrometry-based matrix-assisted laser desorption/ionization (MALDI) and
electrospray ionization (ESI) have been the most popular MS methodologies used for
protein and peptide identification in proteomics (Newton et al., 2004). Other strategies
have included, for example, quadrupole ion-trap (LCQ) and Fourier transform ion
cyclotron resonance (FTICR). MS-based techniques for the separation and sequencing of
peptides are usually a bbottom-upQ approach, complementary to upstream electrophoretic
or chromatographic methods (for the separation of intact proteins) and digestion of
proteins to smaller peptide fragments (Jensen, 2004). This bbottom-upQ tactic to
proteomics has proved highly flexible and versatile. Intact-protein btop-downQ
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approaches, where individual proteins are selected for MS analysis without any previous
chemical or enzymatic proteolysis, have also been used in some situations as a means to
complement peptide-level measurements with more information on the protein
modification state. However, not all intact proteins are amenable to this btop-downQ
methodology mainly due to intrinsic factors related with low solubility and molecular
weight (Jacobs et al., 2005, Loo et al., 2005, Fountoulakis and Vlahou, 2005).
Fig. 1. Classification of affinity ligands for the purification of proteins by affinity chromatography. Biospecific
ligands are naturally occurring molecules with affinity for the target protein-binding site. Pseudobiospecific
ligands can be biological or non-biological molecules that interact with the target protein but do not occur
naturally in the biological systems. Biomimetic ligands are a new class of pseudobiospecific ligands that are
tailor-made to a specific target protein in order to increase specificity and the overall operational performance of
the affinity adsorbents. HCIC — hydrophobic charge induction chromatography; IMAC — immobilized metal
affinity chromatography.
problematic by diluting the sample during the washing step and also by losing low-
abundance proteins if they present affinity for the immobilized ligands. Another option
is the concentration/extraction method, where affinity chromatography can be helpful
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chromatography of proteins (Fig. 3). Three main design strategies have been followed: (i)
the template for design is a natural partner involved in binding to the target protein (Li et al.,
1998); (ii) the template for design is a molecule that binds to the target site of the protein
(Teng et al., 1999; Filippusson et al., 2000); and (iii) direct mimicking of natural biological
recognition interactions (Palanisamy et al., 1999, 2000) (Table 1). Although it is possible
to rationalise several factors in the ligand design, numerous indefinite factors are
introduced upon immobilization on the solid support: the matrix and the coupling
chemistry in addition to the ligand play an important role in binding to the target molecule
(Lowe, 2001). However, performing the synthesis and screening on the solid-support helps
to minimise these effects when considering the final application of the ligand as an affinity
adsorbent.
Fig. 3. General strategy followed for the finding of a lead ligand following the design, synthesis and screening of
a solid-phase combinatorial library of de novo designed ligands. Different strategies can be applied at the
molecular modelling stage of de novo design of synthetic ligands (DESIGN), followed by the synthesis of a nxn
combinatorial library of immobilized ligands (SYNTHESIS) further assessed for affinity and specificity for the
target proteins (SCREENING). Screening procedures can involve, for example, FITC-based systems (a) or
affinity chromatography (b). Potential lead ligands selected at this stage are synthesized in solution-phase,
characterized by NMR and MS and further immobilized onto agarose beads to confirm affinity for target protein.
A lead ligand is chosen and subsequent studies were performed to optimise its performance. A second generation
of ligands might be designed, synthesized and tested.
8
Table 1
A summary of de novo designed biomimetic ligands based on a triazine-scaffold for the affinity purification of proteins
Design strategy Synthesis References
Kallikrein Complex kallikrein/kininogen; ligands Solution-phase synthesis (Burton, 1992)
designed to mimic Phe–Arg dipeptide
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inhibitor; ligands designed to mimic (12 ligands)
the natural inhibitor
IgG (Fab fragment) Complex PpL–IgG; ligands designed Solid-phase combinatorial chemistry (Roque et al., 2004b, 2005a,
towards mimicking the natural PpL (169 ligands) 2005b)
binding interfaces to IgG
IgG Complex SpA–IgG; ligands designed Solution-phase synthesis (Li et al., 1998)
to mimic the Phe132–Tyr133 dipeptide
on SpA
IgG Refinement of SpA mimicking ligand Solid-phase combinatorial chemistry (Teng et al., 1999, 2000)
(Li et al., 1998) (88 ligands)
Human recombinant factor VIIa Complex tissue factor/factor VIIa; Solid-phase combinatorial chemistry; (Morrill et al., 2002, 2003)
ligands designed to bind to the solution-phase synthesis of a sub-library
Gla-domain in Factor VIIa
Sugar moieties on glycoproteins Protein–carbohydrate complexes: Solid-phase combinatorial chemistry (Palanisamy et al., 1999, 2000;
identification and mimicking of key (80 ligands) Gupta and Lowe, 2004)
residues determining monosaccharide
specificity
Recombinant insulin precursor MI3 Study of the target protein per se and Solid-phase combinatorial chemistry (Sproule et al., 2000)
selection of appropriate binding sites (64 ligands); solution-phase synthesis
of a sub-library
Human a1-antitrypsin Study of the target protein per se and Solid-phase combinatorial chemistry (Lowe et al., 2001)
selection of appropriate binding sites
Prion protein Study of the target protein per se and Solid-phase combinatorial chemistry (Renou et al., 2004)
selection of appropriate binding sites (49 ligands)
SpA — staphylococcal protein A; PpL — protein L from Peptostreptococcus magnus; IgG — immunoglobulin G; Gla — g-carboxyglutamic acid.
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From a proteomics point of view, de novo designed synthetic ligands represent a good
alternative to biological ligands and an opportunity to attain new classes of ligands for
specific target proteins, as the power of designed ligands lies on the btailor-madeQ
approach. As already presented in Table 1, the concepts of de novo designed synthetic
ligands has already lead to the development of a series of triazine scaffolded molecules
presenting affinity to different proteins or families of proteins. The utilisation of affinity
chromatography as a depletion method has been recently reported to eliminate the
immunoglobulin content from crude samples to be further submitted to 2D-PAGE or MS
techniques (Lee and Lee, 2004). Similarly, affinity chromatography has also been utilised
recently to separate, in a concentration mode, glycoproteins and glycosylated peptides
resulting from proteomic analysis (Hashim et al., 2001; Hirabayashi et al., 2001). In the
former, both general synthetic ligands (e.g. Cibacron blue F-3GA) and immunoadsorbents
(e.g. bovine IgG-specific IgG immobilized on Sepharose) were utilised; in the latter,
lectins were utilised as the ligand molecule. This section will focus particularly on the
work accomplished in our group regarding the design, synthesis, evaluation and
application of (i) immunoglobulin-binding ligands and (ii) glycoprotein-binding ligands
(Fig. 4). Although displaying high selectivity, adsorbents based on proteins A/L and
carbohydrate-binding systems suffer from high costs of production and purification, low
binding capacities, limited life cycles and low scale-up potential, which is attributable to
the biological nature of the ligands. The biomimetic ligands described below are fully
Fig. 4. De novo designed affinity ligands for mimicking proteins A and L. Crystallographic structure of the
complex between protein A and Fc fragment of IgG (A) and of the complex between protein L and Fab fragments
of IgG (D). Structure of the ligands artificial protein A (B) and L (E) and respective superimposition at the
proteins A and L binding sites (C) and (F).
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synthetic in nature and can circumvent problems associated with biological ligands, while
maintaining the affinity and specificity for the target proteins.
Staphylococcus aureus protein A (SpA) was used as a template for the design of a
synthetic ligand to bind specifically to immunoglobulin G (Li et al., 1998). The dipeptide
motif Phe132–Tyr133 was crucial for the complex SpA–IgG (Deisenhofer, 1981) and was
thus used as a starting point for the design of a series of biomimetic ligands. The most
suitable ligand was able to bind IgG selectively from human plasma (98% purity after
elution) and competitively to SpA, with affinity constants between 105–106 M 1 (Li et al.,
1998). The lead compound was further refined by the synthesis of an IgG-binding
combinatorial solid-phase ligand library comprising 88 adsorbents. The ligand library was
assessed for its ability to bind pure human IgG and selected ligands further tested for their
specificity to purify IgG from human plasma (Teng et al., 1999). Ligand 22/8 (ApA—
artificial protein A) proved to be the most efficient for binding hIgG, (K a = 1.4 105 M 1)
and for the separation of IgG from human plasma with recoveries of 67–69% and purities
of 98–99% (Teng et al., 2000). This adsorbent also proved effective in the purification of
antibodies from different species (chicken, cow, rabbit, pig, horse, rat, goat, sheep and
mouse) and from different human classes (IgA and IgM) and IgG subclasses, including
IgG3. Stability studies of ligand 22/8 in 1M NaOH over N140 h proved that it can
withstand general procedures of cleaning-in-place and sterilisation, which represents an
important advance over natural SpA (Teng et al., 2000).
The lack of existence of a synthetic affinity ligand with a universal affinity for the scFv,
Fab or F(abV)2 small antibody fragments prompted the design, synthesis and evaluation of
a protein L mimic. Until then, affinity ligands binding specifically to the Fab fragments of
immunoglobulins comprised anti-antibodies raised against the immunoglobulin of interest,
molecules interacting with CDR regions (antigen or epitope/mimotope peptides) or
microbial binding proteins (protein L). However, anti-antibodies represent an expensive
and labour intensive option (Huse et al., 2002); ligands interacting with CDR regions are
usually unique to each antibody and, therefore, do not represent a universal ligand (Murray
et al., 1998). Protein L from Peptostreptococcus magnus was discovered in 1985 and is an
immunoglobulin light chain binding protein particularly suitable for the purification of
scFv, Fab and F(abV)2 biomolecules (Housden et al., 2003). Protein L binds with high
affinity (K d of 1n M) to a large number of immunoglobulins with n1, n3 and n4 light
chains (but not to n2 and E subgroups) and thus recognises 50% of human and more than
75% of murine immunoglobulins (Stura et al., 2002).
A solid phase combinatorial triazine-scaffolded library of affinity ligands was designed
de novo, synthesized and screened for the discovery of a protein L mimic. The thirteen
compounds included in the combinatorial library were selected on the basis of the structure
of the eleven different amino acid residues from the protein L domain recognised as being
involved in the interaction with the n-light chains (Wikstrom et al., 1995; Graille et al.,
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glucose oxidase with a K a in the order of 4.3 105 M 1 (Palanisamy et al., 2000). This
biomimetic ligand displayed affinity for mannose, glucose and fucose moieties and the
ability to bind to glycoproteins but was unable to bind selectively to the different
saccharides (Palanisamy et al., 2000). Rational design and combinatorial chemistry were
further employed on the discovery of ligand 11/11, a benzylamine bis-substituted triazine
molecule, showing the following monosaccharide specificity mannoside N glucoside N
galactoside, and affinity constants for binding to glycoproteins in the order of 104 M 1
(Gupta and Lowe, 2004). This biomimetic ligand also enhanced the understanding of
saccharide selectivity, showing that the resolving power might be based on the formation
of hydrogen bonds between the equatorial 3- and 4-hydroxyl groups from sugars and the
planar, polar nitrogen groups of the triazine ligand. Ligand 11/11, like other triazine-based
biomimetic ligands, has shown to be chemically stable and easy to synthesize, to possess
non-fissile bonds and to withstand sterilization and cleaning in situ (Gupta and Lowe,
2004).
5. Concluding remarks
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