Affinity Interactions by Capillary Electrophoresis: Binding, Separation

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Affinity Interactions by Capillary Electrophoresis: Binding, Separation, and Detection Fangzhi Yu, Qiang Zhao, Dapeng Zhang, Zheng Yuan, and Hailin Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04741 • Publication Date (Web): 04 Nov 2018 Downloaded from http://pubs.acs.org on November 5, 2018

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Analytical Chemistry

Affinity Interactions by Capillary Electrophoresis: Binding, Separation, and Detection Fangzhi Yu1,2, Qiang Zhao1, Dapeng Zhang1, Zheng Yuan1,2 Hailin Wang1,2,* 1. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China 2. University of Chinese Academy of Sciences, Beijing, 100049, China

* Corresponding Author Email: [email protected] Tel/Fax:+86-10-62849600

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Contents 1. General introduction 2. Instrumentation 3. Methodologies and technologies 3.1 Antifouling coating for elimination of capillary wall adsorption 3.2 Chemical crossing-link facilitates the measurement of protein-protein interactions 3.3 Preservation of protein activity by temperature-controlled ionic liquid aqueous two phase systems 3.4 Minimization of Joule heating for measuring temperature-sensitive affinity interactions 3. 5 Supramolecular receptor for CE separation of modified peptides 4. Theoretical study of CE-based binding measurements 4.1 Moment analysis-based affinity CE 4.2 Theoretic and experimental improvement on CE-frontal analysis 5. Affinity and kinetic measurement 5.1 Multivalent interactions and cooperative assembly 5.2 Tandem of inline reaction and CE separation 6. Aptamer-based affinity capillary electrophoresis 6.1 Nucleic acid library and aptamer selection 6.2 Extended sequence for enhancing affinity of aptamer 6.3 Sensitive and specific assay for detection of target molecules 7. Capillary electrophoresis immunoassays 8. Chiral selector and CE-based chiral analysis 9. Summary and perspectives 2


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1. General introduction Affinity interactions between molecules constitute this amazing and mysterious world of the human-living earth. At the level of individuals, affinity interactions are fundamental for the paramount functions and exquisite architectures of all organisms in life, for the occurrence and development of diseases, for the therapy-associated diagnosis, pharmaceutics and medicine (and cutting-edge regenerative medicine), and for the environment-life interplayed sphere. At the level of cells, affinity interactions function critically throughout every facet of every cell (e.g., division, differentiation, and motility), between cells (e.g., communication, aggregation, and immune response), and during reproduction, organogenesis and organ maintenance. At the level of molecular events, affinity interactions allow the cells to make DNA package (e.g., nucleosome, chromosome), to perform precise DNA replication and timely DNA repair, transcription, translation, mRNA splicing, and to modify proteins and nucleic acids (typically in epigenetics). In essence, all molecules within and outside the cells, such as proteins, nucleic acids, hormones, and nutrients play vital roles through affinity interaction network in dictating and regulating fundamental life processes.

A number of techniques, including isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), fluorescence spectroscopy, (cryo-)electron microscopy, and capillary electrophoresis (CE), have been developed to measure the affinity interactions. Each technique has its own advantages and limitations. Noteworthily, CE as a powerful and popular technology can be used to measure the affinity interactions at the level of molecules (DNA, RNA, proteins, chemicals, and etc.), at the level of single cells, and between cells, probably individual organisms (e.g., bacteria, zygotes and early embryo). This is unprecedented compared to other known technologies. These strengths benefit from the anticonvective properties of small internal diameter tubes (10 m – 200 m) routinely used for CE, which allows the efficient separation-based 4


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resolution of bound and unbound species through the high electric field with dramatically reduced re-mixing of the unbound and bound species. It provides rapid measurement of affinity interactions in free solution or aqueous solution containing linear polymer or crosslinked gel network. Because of rapid and aqueous solution-based separation, least perturbation on the target interactions is introduced in the process of CE study. Moreover, there are a number of formats can be adapted to diverse interaction studies as reviewed in one recent article.1 CE can provide more specific and accurate binding details (e.g., interaction sites, binding stoichiometry, and binding constants), which are helpful to better understand the molecular mechanisms of the basic processes. More importantly, CE determination can be performed under physiologically relevant conditions, which makes it very suitable for studies of biomolecular interactions.

As described above, CE can be used for measuring molecular interactions, but it can also utilize the molecular interactions to facilitate separation for analysis of target analytes or to improve the detection specificity. In this review, we highlighted recent two-year advances (2017-2018) in instrumentation, theory, methodologies and technologies of CE for measuring affinity interactions and for applying affinity interactions to perform specific and sensitive identification, screening and analysis.

2. Instrumentation CE provides efficient separation of bound and unbound species involved in the affinity interactions, however, it could not directly generate any signal on these separated species. Therefore, to exploit the interactions, it must be coupled with a signal-generating and receiving detector. Essentially, CE is very flexible and can be coupled with various detection technologies, typically, ultraviolet-visible spectrometry (UV-vis), fluorescence (in particular, laser-induced fluorescence), mass spectrometry, and so on.

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The coupled laser-induced fluorescence (LIF) provides extremely high sensitivity for the CE-based interaction study. It is prerequisite that one of the binding partners is fluorescently labeled. Interestingly, the unique polarization (anisotropy) property of fluorescence, which reflects the molecular size-correlated diffusional rotation rate of molecules or the complexes, called fluorescence polarization (FP), has been utilized in the CE-LIF to form an extraordinary useful CE-LIFP technique for the study of immunereactions and affinity interactions and for immunoassays and aptamer affinity analysis. Essentially, the online coupled FP facilitates the confirmation of the identified interaction as revealed by the CE-driven mobility shift without performing additional experiments, and also facilitates the identification of binding stoichiometry and unresolved unboundand bound-species. Noteworthily, we showed that CE-LIFP could be used to monitor the DNA wrapping around protein,2 and to map the protein-nucleic acid interaction at single nucleotide resolution.3

To improve the throughput of the CE-LIF, Woo et al. built up a CE coupled with dual color LIF (Figure 1).4 This instrumental setup allows the simultaneous detection of thyroxine (T4), triiodothyronine (T3), and thyroid-stimulating hormone (TSH) in a single run of CE for enhancing diagnosis of thyroid gland disease. Interestingly, they also showed that the complex of antibody-T4 could be efficiently separated from the antibody and the antibodyTSH complexes although the molecular sizes of these species mainly determined by the antibodies are similar. Similarly, the antibody-TSH complexes could be separated from the antibody, too.

The hyphenation of CE and molecular structure-deciphering mass spectrometry (MS) provides unambiguous information on the binding partners and the related binding stoichiometry.5-7 However, it is difficult to measure the binding constants of biomolecular interactions accurately due to its incompatibility to the aqueous solution containing 6


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inorganic salts of mM in concentrations, which are often required for physiologically relevant interactions. For an instance, Qian et al. exploited electrospray ionization (ESI)MS to screen the binding of small molecules with oligo DNA by the variation in stoichiometry, but they measured the binding constants using CE-UV based frontal analysis (FA) rather than MS detection.7 Interestingly, the use of CE-MS can simultaneously characterize and quantify the binding of metal-based anticancer agents (cisplatin and the organoruthenium RM175) to an oligodeoxynucleotide (5’dATTGGCAC-3’and ubiquitin (Figure 2). Moreover, for the first time, oligonucleotide metallation was resolved at single-nucleotide resolution by MS.8

CE can also be hyphenated with element-sensitive inductively coupled plasma mass spectrometry (ICP-MS), providing a powerful means for measuring the interactions involved with any metal or metalloid element. By the means of CE-ICP-MS, it is possible to explore the fate of differently functionalized gold nanorods (AuNRs) in human serum. As an in vitro study,9 it was reported that apo-transferrin (rather than abundant albumin) dominates the protein corona of carboxyl-modified AuNRs. In contrast, the albumin conjugate forms “soft” protein corona of the AuNRs with surface amino-groups, exhibiting higher affinity toward aminated surface. However, it becomes slowly replaced by other, less abundant proteins.

3. Methodologies and technologies 3.1 Antifouling coating for elimination of capillary wall adsorption In spite of many merits of CE, it is often observed the adsorption of analytes (e.g., proteins) on the inner wall of routinely used fused-silica capillary. At least two side effects arise from the protein adsorption: 1) Causes peak broadening, low separation efficiency, and poor reproducibility. When the adsorption becomes very serious, no target protein analyte migrates out from the capillary; 2) Disturbs the target biomolecular interactions. A number 7



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of strategies have been developed to minimize protein-wall interactions, including capillary derivatization,10 extreme pH,11 surfactant additives,12 and high ionic strength buffers.13 Recently, an antifouling strategy was developed for alleviating the protein adsorption on the capillary inner wall. By modifying the capillary with poly(2-methyl-2oxazoline) (PMOXA) and its derivatives, the modified capillary exhibits similar antifouling capacity and even better stability compared with the gold standard poly(ethylene glycol) (PEG).14-16 Star-shaped polymer could be synthesized for diverse applications.17 Du et al. reported a modification of fused silica capillary with star-shaped poly(2-methyl-2-oxazoline)-based copolymer,18 forming the antifouling capillary. In this case, the star-shaped copolymer was formed by being grafted with poly(ethylene imine) (PEI) and then immobilized onto the fused-silica capillary inner wall via dopamine-assisted co-deposition strategy. The separation of a basic protein mixture (cytochrome c, lysozyme, ribonuclease A and α-chymotrypsinogen A) is greatly improved, showing about ten-fold higher separation efficiency over those obtained with a bare capillary. Moreover, the reproducibility of the migration is also improved with the relative stand deviation (RSD) values of migration time less than 0.7% (30 consecutive runs). This modified capillary is successfully used to study the protein-drug interactions via frontal analysis. Of note, the modified capillary shows negligible electroosmotic flow (EOF) at pH 3.0-10.0. The same group also developed another type of modification of the capillary using poly (2-methyl2-oxazoline)-random-glycidyl methacrylate (PMOXA-r-GMA) copolymer.19 Of note, the modified capillary shows stable EOF at pH 2.2 -9.0. Interestingly, it also exhibits excellent separation efficiency for the basic proteins.

3.2 Chemical crossing-link facilitates the measurement of protein-protein interactions The interactions occurred between diverse proteins exhibit varying affinity spanning a wide range of dissociation constant, KD (M – nM), and varying binding stoichiometry (1:1 to very high order, e.g., protein-assembled nano-machine). Moreover, to use the CE method for probing the protein-protein interactions (PPI), it often suffers from two dilemmas: 1) 8


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The adsorption of proteins onto the inner wall of capillary; 2) instability of protein-protein complexes during CE separation, resulting in the dissociation of the protein-protein complexes. Most techniques used for minimizing protein adsorption to the capillary are often not helpful to maintain noncovalent protein interactions. We reported that the use of serum albumin or IgG as a sample buffer additive can reduce the protein adsorption and stabilize the protein-DNA complexes, enhancing the immuno-detection of DNA adducts.20 However, it remains a challenge to maintain non-covalent PPIs with weak affinity and fast dissociation kinetics during CE separation. Kennedy group reported the protein cross-linking capillary electrophoresis (PXCE) to overcome these limitations.21 In PXCE, the proteins are cross-linked under binding conditions and then separated. They first tested formaldehyde as a cross-linking agent, and found that PXCE could determine the KD values of three protein−protein complexes: the complex of lysozyme−antilysozyme, Hsp70−Bag3 heterodimer, and heat shock protein 90 (Hsp90) homodimer. Without the use of the cross-linking agent formaldehyde, the complex of lysozyme−antilysozyme and the Hsp70−Bag3 heterodimer could not be detected, and the Hsp90 homodimer was reluctantly detected. By the use of PXCE, they found that lysozyme-antibody interaction has a KD value of 24 nM, and Hsp70−Bag3 heterodimer has a KD value of 25 nM. They also applied PXCE to quantify inhibition of PPIs with Hsp70−Bag3 binding site mutants and small molecule inhibitors. To improve cross-linking between interacting protein partners, later, the same group exploited glutaraldehyde as the cross-linking agent.22 Consistent with rapid reaction kinetics of glutaraldehyde, it took only 10 s for glutaraldehyde to complete cross-linking, but 10 min for the formaldehyde to do so. They further expanded the utility of PXCE to assess a wide range of PPIs (Figure 3). These PPIs involved with weak and multimeric oligomers. The former could not be detected by the conventional CE due to their dissociation, and the latter could not be differentiated between the complexes of distinct stoichiometry due to the requested mild conditions for preservation of the complexes during CE separation. The cross-linking of the complexes allows the possibility to use interaction-sensitive but separation-favorable CE conditions. They quantified seven 9



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different PPIs (KD: 3.8 μM to 2.1 nM), including Hsp70-heat shock organizing protein (3.8 ± 0.7 μM) and bcl2-anthanogene (26 ± 6 nM). They also assessed non-specific crosslinking of protein aggregates using size exclusion chromatography, and found that it was minimal at protein concentrations

Affinity Interactions by Capillary Electrophoresis: Binding, Separation

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