Ionic liquids in the assay of proteins

Abstract

As a group of novel green solvents, ionic liquids have attracted extensive attention and gained popularity in various applications including protein assays. Ionic liquids are not only an excellent reaction medium to replace the conventional organic solvents, but are also an efficient participant to achieve better performances. The aim of the present mini-review is to illustrate the state-of-the-art progress of implementing ionic liquids in protein assays, focusing on the investigations of protein stability/activity, protein extraction and isolation/purification, protein crystallization, separation of protein species and their detections.

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Introduction

As a group of organic salts consisting entirely of ions (anions and cations), ionic liquids (ILs) exist in the form of a liquid at a low temperature (<100 °C). Recently, ionic liquids have shown great promise as a potential alternative to conventional volatile toxic organic solvents due to their unique and attractive properties including negligible vapor pressure, nonflammability, high chemical/thermal stability, low toxicity and favorable conductivity. They have also been defined as “designer solvents” for the fact that the physicochemical properties of ionic liquids such as their viscosity, hydrophobicity/hydrophilicity, polarity and miscibility can be controlled by flexible selections of suitable ionic components and the attached substituents to customize the ionic liquids in order to fulfil specific demands. These attractive features of ionic liquids have thus made them popular candidates in a wide number of applications in various fields including organic synthesis, extraction/separation, electrochemical analysis, catalysis and chemical sensors etc. During the past few years, a number of reviews concerning the physicochemical properties and the applications of ionic liquids have witnessed their booming popularity.^1–5

Protein assay has long been a crucial issue in biosciences as well as some of the related fields. In this respect, the applications of ionic liquids, i.e., the environmentally benign solvents, to perform protein assays have received increasing attention in the past few years. This can be attributed to the fact that ionic liquids not only provide a novel and highly efficient reaction medium, that is the solvent, but they also serve as efficient participants in the various chemical/biological reaction processes. Fig. 1 illustrates the number of publications per year in the last decade concerning the applications of ionic liquids related to the assay of proteins. It can be seen that the investigations in this field are still in their infancy characterized by the limited number of publications up to now. However, in the mean time, it shows an obvious increase in the interest on protein assays and the related topics very recently. The insert in Fig. 1 illustrates the distribution of publications in the various aspects of protein assays (in average), e.g., protein stability/activity, protein separation, protein extraction and purification, protein crystallization and detection. Some pertinent details are discussed in the following sections.

Fig. 1 The number of publications per year concerning the applications of ionic liquids related to protein assays in the last decade. The insert denotes the distribution of publications in the various aspects of protein assays (in average) (Source of information: ISI Web of Knowledge).

Protein stability/activity in ionic liquid phase

The studies of protein folding are normally carried out in diluted aqueous solutions in order to avoid protein aggregation, and the exploitation of a renaturation additive preventing aggregation and enabling the recovery of protein activities at high concentrations is generally highly demanded. Summers and Flowers investigated protein refolding by exploiting ionic liquids as additives. The adopted ionic liquid, i.e., ethylammonium nitrate (EAN), effectively suppressed the aggregation of lysozyme and gave rise to a significant increase in the refolding yields at a lysozyme concentration up to 0.5 mol L⁻¹.⁶ It is assumed that the interactions between the cationic moieties of the ionic liquid and the exposed hydrophobic regions of the protein molecules induced by high temperature help to prevent the aggregation of protein. Afterwards, the thermostability of different protein species such as lysozyme,^7–10 cytochrome c (cyt-c),¹¹ human serum albumin,^12,13 ribonuclease A,^14,15 lipase B^16,17etc. in various ionic liquids have also been investigated and the results all implied that ionic liquids are useful and efficient additives for the effective suppression of protein aggregation. The intrinsic emission monitoring of tryptophan residue of protein also indicated that the use of ionic liquids produces an astonishing entropically driven stabilization in opposition to the thermal unfolding, i.e., the unfolding entropy, ΔS, is determined to be 250 and 136 J K⁻¹ mol⁻¹ for monellin in water and in ionic liquid [bmpy][NTf₂] respectively. This observation is consistent with the more rigid solvation behavior in the ionic liquid phase.¹⁸

Handling and storing of proteins in biocompatible ionic liquids bring forward obvious advantages over that in the conventional aqueous buffer solutions. Lysozyme stored in ionic liquid choline dihydrogen phosphate (dhp) after one month still possess higher activity and fraction of folding than those obtained for lysozyme stored in aqueous buffer.¹⁹ This ionic liquid is also able to serve as a strong stabilizing medium for cyt-c offering storage periods up to 18 months without the loss of protein activity.^20,21 As a comparison, cyt-c is prone to becoming inactive after only one weeks storage in a conventional aqueous buffer solution.

Further investigations by differential scanning calorimetry (DSC), fluorescence and circular dichroism (CD) suggest that the improved thermostability of proteins in the ionic liquid phase might result from the alteration of hydration level and structural compaction in the protein molecules of interest.^18,22 Yet up to now, the exact explanations for the increased renaturation and thermostability of proteins in the presence of ionic liquids have not been well understood and the underlying interpretations are still mostly speculative. So far, many of the factors might be treated as potential contributors,⁷ these include polarity, hydrogen bond basicity, anion nucleophilicity, the framework of the ionic liquids, the ion's kosmotropicity and viscosity. However, none of these properties are solely responsible for the functions of proteins in the ionic liquid phase. In many cases, multiple factors have to be considered, i.e., the investigation of lipase B activities in more than twenty ionic liquids has indicated that the protein stability/activity induced by ionic liquids might be the result of a comprehensive contribution from the ionic liquid's polarity and viscosity, hydrophobicity, ionic association strength, hydrogen-bonding and protein dissolution etc.¹⁶

Protein crystallization in the presence of ionic liquids

X-ray and neutron diffraction analyses are widely adopted techniques used to acquire conformational information about protein species in bioscience. In this respect, the availability of high-quality protein crystals with suitable sizes is generally an indispensable prerequisite. The application of ionic liquids in the crystallization of protein was first reported by Garlitz et al. with EAN acting as a precipitating agent. Their experiments produced large clumps of lysozyme crystals resembling starbursts and single rectangular lysozyme crystals suitable for diffraction analysis, and the crystallographic refinements indicated that the obtained lysozyme crystal maintains its native structure.²³ Afterwards, the performances of ionic liquids comprising different cations (imidazolium, phosphonium, pyrrolidinium, ammonium, etc.) and anions (borate, halide, sulfate, acetate, glycolate, nitrate, phosphate, etc.) were employed to investigate the protein crystallization process. Judge et al. demonstrated that proteins (lysozyme, catalase, myoglobin, trypsin, glucose isomerase, and xylanase) crystals grown with ionic liquids as precipitants or additives both provide X-ray diffraction resolution similar to or better than that obtained without ionic liquids. In addition, the performances of the ionic liquids are more prominent when used as crystallization additives than precipitation agents.²⁴ Considering that crystallization stems from the interfacial effects of strongly hydrated anions near the polar surface of the proteins species,²⁵ ionic liquids used as crystallization additives result in a favorable fine tuning of the interfacial effects near the surface of the proteins facilitating their crystallization and the tuning abilities of ionic liquids with strongly hydrated anions are superior to those with weakly hydrated ones.^26,27 Very recently, the direct crystallization of lysozyme from egg white was achieved by using hydrophilic ionic liquid 1,3-butylimidazolium chloride as an additive.²⁸ The elevated threshold to super-saturation of lysozyme induced by the negligible vapor pressure of ionic liquid provides a controlled velocity for the growth of lysozyme crystal. These eventually promote the crystallization of lysozyme, i.e., less crystal polymorphism and precipitation while larger crystals and significantly improved the tolerance to the concomitant impurities or sample matrices. Therefore, it enables direct crystallization of lysozyme from complex sample matrix, i.e., egg-white, which opens up a promising avenue for the development of protein crystallization methodology and offers potential for the practical separation/purification of proteins from complex sample matrices.

There is no doubt now that the addition of suitable ionic liquids into the protein crystallization medium promotes the crystallization process. The precipitation of protein could be effectively avoided even at higher salt concentrations, and the crystal polymorphism is reduced. At the same time, lager sized and better shaped protein crystals could be obtained.^26–30 The negligible vapor pressure of ionic liquids is considered to be among the main contributions to the improvement of protein crystallization.^29,30 The addition of ionic liquids into the protein solution would certainly slow down the rate of vapor transfer rate and therefore enable a slower dynamic approach to super-saturation and controlled crystal growth velocity, which are favorable to the nucleation and crystal growth regime resulting in crystals under fixed crystallization conditions. Another potential mechanism for the positive effect of ionic liquids on protein crystallization is attributed to the increased protein solubility in the presence of ionic liquids,²⁹ which means that it is even difficult to reach a super-saturation state, or it provides a lower super-saturation degree. This is beneficial to the avoidance of protein precipitation or amorphous aggregation.

Ionic liquids in protein separations

Capillary electrophoresis (CE) is a powerful technique for protein separation. In practical applications, the adsorption of proteins on the native capillary surface induced by electrostatic interactions is very annoying and not only results in broadened bands but also generates low protein recovery. The Jiang's and Corradin's groups demonstrated that the dynamical coating of capillary with imidazolium-based ionic liquids provides an efficient approach to suppress protein adsorption due to the successful surface charge reversal on the capillary wall and the generation of an anodic electroosmotic flow,^31,32 as illustrated in Fig. 2. When the ionic liquid-modified CE systems were employed for the separation of basic proteins, i.e., lysozyme, cyt-c, trypsinogen and α-chymotrypsinogen, fast and efficient separation was achieved along with symmetrical peaks.

Fig. 2 Schematic of mechanisms for the separation of proteins by using imidazolium-based ionic liquids. (Adapted from Ref. 31, with kind permission from Elsevier Science Publishers.)

1-Butyl-3-methylimidazolium tetrafluoroborate has been proven to be a useful dynamic coating reagent and running electrolyte for CE separation of both basic and acidic protein species giving rise to satisfactory results.³³ The dynamic coating has been demonstrated to be stable for over 120 h without any regeneration between runs. At the same time, ionic liquids as running electrolytes provided better ability to maintain the background current when compared to common electrolytes.

With an ionic liquid as an additive in the running buffer, selective sample injection in the CE process could be achieved owing to the high conductivity of ionic liquids. In practice, the actual voltage for sample injection was greatly increased, leading to a field-amplified sample stacking effect favorable for the enrichment of positive analytes at electrokinetic injection mode. This technique was well demonstrated in the enhanced detection of avidin with CE-electrochemiluminescence by using [Bmim][BF₄] as CE selectors. A detection limit of 12.5 nmol L⁻¹ is achieved, which is an improvement of over one order of magnitude compared to that achieved in the absence of an ionic liquid.³⁴

A hydrophilic ionic liquid, i.e., 1-butyl-3-methylimidazolium dodecanesulfonate (BAS) has been designed and prepared. It has been successfully applied as a supporting electrolyte and surface modifier in a microchip-based micellar electrokinetic chromatographic system for protein separation.³⁵ The exploitation of BAS in microfluidic systems not only offers a guaranteed ionic strength to enhance the electroosmotic flow, but also effectively eliminates the adsorption of proteins on the surface of the PDMS microchannels.

Protein detection with ionic liquids as matrices

A class of special ionic liquids formed by commonly used matrix-assisted laser desorption/ionization (MALDI) acidic matrices with organic bases have been designed as suitable matrices for mass spectrometric (MS) detection. These have been termed as “ionic liquid matrices” (ILMs) to distinguish from conventional ionic liquids. Armstrong et al. first found that ILMs not only produce a much more homogeneous sample solution as all the liquid matrices do, but also provide a greater vacuum stability than most of the solid matrices.³⁶ They have also revealed that only those ILMs showing significant absorbance at the desired wavelength and with protons available are effective matrices for MS analysis, whereas most conventional ionic liquids are not quantified at this point.

Various ILMs have been investigated for the MS analysis of biomolecules, these include 2,5-dihydroxybenzoic acid butylamine, R-cyano-4-hydroxycinnamic acid butylamine, 3,5-dimethoxycinnamic acid triethylamine, and solid MALDI matrices 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid.³⁷ The results indicated that 3,5-dimethoxycinnamic acid triethylamine is the best ILM for the detection of proteins of high molecular weight, such as IgG, which facilitates protein identification by yielding higher scores and increased sequence coverage compared to commonly used MALDI matrices. Cramer and Coreless also showed that, compared to conventional crystalline matrices, the ILM consisting of α-cyano-4-hydroxycinnamic acid and N,N-diethylamine led to improved identification of the model protein myoglobin.³⁸ In addition, the use of ILMs significantly reduces unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices. In particular, matrix and low-mass ion signals and ion signals resulting from the formation of cation adduct are dramatically reduced. As a result, the improvement of signal-to-noise ratios and spectral quality in terms of reduction of matrix clusters and chemical noise are achieved.^39,40

Protein extraction into ionic liquid phase

The direct extraction of protein species of interest from aqueous medium into an appropriate ionic liquid phase has been considered to be not practical, this is attributed mainly to the fact that proteins are insoluble or have very limited solubility in the ionic liquid medium. The first attempt for the direct extraction of proteins by ionic liquids was reported by Wang's group.^41,42 Heme-proteins, i.e., hemoglobin and cytochrome-c were quantitatively extracted into a hydrophobic ionic liquid 1-butyl-3-trimethylsilylimidazolium hexafluorophosphate ([Btmsim][PF₆]). Both the ⁵⁷Fe Mossbauer spectra and CD spectra indicated that the interactions between the iron atom in the heme group of hemoglobin and the cationic ionic liquid moiety, i.e., the imidazolium cation, furnishes the main driving force for the transfer of hemoglobin into the ionic liquid phase.

On the contrary, the extraction of proteins by ionic liquid in the presence of proper assistant extractants or additives is more popular because the existence of assistant extractants help to enhance the solubility of the proteins of interest. Shimojo et al. first reported the extraction of heme proteins from an aqueous phase into the ionic liquid [C_nOHmim][Tf₂N](n = 2,4,6,8) with dicyclohexano-18-crown-6 (DCH18C6) as an assistant extractant. The enhanced solubility of cytochrome-c in the ionic liquid phase induced by the coordination between DCH18C6 and the lysine residues of proteins well facilitates the extraction process,^43,44 as showed in Fig. 3. Another efficient way to overcome the solubility limitation is to form an aqueous domain, i.e., stable water droplets (e.g. reverse micelles) in the ionic liquid's continuous phase, to enable the dissolution of proteins.⁴⁵ A water-in-ionic liquid reverse microemulsion, i.e., the water/AOT/1-butyl-3-methylimidazolium hexafluorophosphate reverse microemulsion system, has been demonstrated to entail selective extraction of hemoglobin with an extraction efficiency of 96%. This extraction system was used for the successful isolation of hemoglobin from human whole blood.⁴⁶

Fig. 3 Schematic illustration of the extraction of Lys-rich protein in an aqueous medium to an ionic liquid [C_nOHmim][Tf₂N] with DCH18C6 as an assistant extractant. (From Ref. 43, with kind permission of American Chemical Society.)

The exploitation of hydrophilic ionic liquids to form aqueous two-phase systems (ATP) is also established as an economical and efficient extraction approach for the extraction of proteins.^47–50 The extraction of various protein species, e.g., serum albumin, trypsin, cyt-c, α-globulins and horseradish peroxidase, with ionic liquid-based ATP systems demonstrates well the extraction efficiency and the practical applicabilities. Recently, an online solid phase extraction system was developed for the direct extraction of bovine serum albumin by using an ATP system consisting of a hydrophilic ionic liquid of 1-butyl-3-methylimidazolium chloride and K₂HPO₄.⁵¹ Low levels of proteins were quantitatively extracted into the ionic liquid rich upper phase with a distribution ratio of ca. 10 between the upper and the lower phase and an enrichment factor of five. A sufficient amount of K₂HPO₄ added into the separated upper phase results in a further phase separation, giving rise to an improved enrichment factor of 20.

Conclusions

The extensive investigations involving ionic liquids demonstrate its vast potential in various fields of biological concern. The present mini-review aims at giving a general overview of what has been done in protein assays. Among the numerous practical applications of ionic liquids, their employment in protein assays is a new field of research just starting to attract attention. In this respect, future studies deserve to be focused on (1) the elucidation of mechanisms concerning the interactions between the ionic liquid moiety and the proteins of interest; (2) the dependence of protein conformations on the structure and property of the ionic liquids; (3) the selective isolation of specific protein species with the aid of ionic liquids; (4) the development of ionic liquid-based probes for the sensitive and selective sensing of proteins. We expect investigations in the above aspects will give rise to further progress in the field.

Acknowledgements

This work is financially supported by the Natural Science Foundation of China (Nos. 20805004, 20635010, the National Science Fund for Distinguished Young Scholars No. 20725517 and the Major International Joint Research Project 20821120292), the Fundamental Research Funds for the Central Universities (N090405004, N090105001) and the State Key Laboratory of Electroanalytical Chemistry (2009003).

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