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Review Article

The Discovery of Low-Abundance Allergens by Proteomics Analysis Involving Combinatorial  Peptide Ligand Libraries

Egisto Boschetti1*,Elisa Fasoli2and Pier Giorgio Righetti2

1Bioconsultantat Jam-Conseil, 92200 Neuilly, France

2Departmentof Chemistry Materials and Chemical Engineering «Giulio Natta», Politecnicodi Milano, Italy

*Corresponding author: Dr. Egisto Boschetti, 92200 Neuilly sur Seine, France,

  Tel: +33 670 523 533; Email: egisto.boschetti@gmail.com

   Submitted: 02-12-2015  Accepted: 02-23-2015  Published:  03-11-2015  

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Article

Abstract

Allergic reactions are biological processes of defense against foreign products that are considered external dangerous agents of various origins. The latter, allergens, modify the expression of immunoglobulins E and G and produce serious reactions that can be life threatening. The increased occurrence of allergic reactions stimulates the investigations around the discovery of novel protein agents, which has been made possible by novel technologies developed for proteomics research where from numerous novel allergens, particularly those of low abundance, have been detected. Many allergens are of high or average abundance in several biological extracts and can be easily identified; others are rare and dilute requiring specific technologies to be first discovered, characterized and then assayed. In this review the focus is centered on low-abundance protein allergens that can only be detectable by using novel enrichment technologies (such has combinatorial peptide ligand libraries, CPLL), which have demonstrated their effectiveness in proteomic discoveries. This technology is thus described with respect to allergen discovery; practical examples of novel allergens from different sources are illustrated. Allergens from plant, from animal source and also those that are part of complex food preparations are discussed.

Keywords: Low Abundance Allergens; Proteomics; Combinatorial Peptide Ligand Libraries; Separation; Enrichment

Introduction

Biological extracts and fluids, whatever their nature, comprise very numerous proteins each of them present at a typical concentration. The difference of concentration between the most concentrate and the most dilute ones can reach extremely high values that in certain instances (e.g. human serum) are estimated close to twelve orders of magnitude [1]. This peculiar situation introduces difficulties in the identification of highly diluted proteins because those classified as of high abundance cover the detection signal. It is because of this major issue that dedicated technologies have been developed. This is the case for instance of the use of adsorbents capable to specifically capture and thus remove the species that are highly concentrated (depletion) evidencing thus proteins that are normally not detectable. Sorbents used in affinity chromatography have been first adopted, but the most powerful are immunosorbents capable of addressing very specifically certain categories of proteins. For instance, when analyzing medium and low-abundance proteins in human serum or plasma it is common to use six [2] or twelve immunosorbents [3] mixed together to remove albumin, IgG, transferrin and a few other abundant proteins. In spite of their declared specificity, non-specific binding is unfortunately always present with the consequence of capturing also many other proteins of interest that escape the following analysis. This is due to several different mechanisms: non-specific binding to the antibody preparations, non-specific binding to the solid matrix where the antibodies are grafted or due to the natural interaction of some low-abundance proteins with removed high-abundance ones.

The losses of many proteins of importance during the depletion process stimulated novel technologies such as the use of solid-phase combinatorial peptide ligand libraries (CPLL) [4]. Under certain conditions they ensure the “compression” of the dynamic concentration range of proteins with the reduction of high-abundance proteins and the concentration of rare species [5] as briefly described in the following section.

The application of CPLL technology is to allow a significant simplification in the elucidation of complex proteomes while digging much deeper compared to current methodologies [6,7]. Another application of such a method is to remove impurities from purified biologicals of therapeutic interest [8] in a unitary single step called “polishing”. The detection of protein traces from beverages (particularly wines, see below) is another very powerful approach with a detection limit that depends only on the volume of the available sample, due to the very large concentration effect of CPLLs.  Within the proteomics investigations CPLLs have been extensively used for evidencing biomarkers of well-targeted diseases including cancers [9,10]. Interestingly, within this domain, the fact that CPLLs are able to  concentrate  the  very  low-abundance species, early-stage potential biomarkers could be identified [11-13].

The detection of proteinaceous allergens has been also implemented with the use of CPLLs as an “amplification” system, in order to see if it was possible to detect polypeptides of low or very low-abundance, recognized by IgE of allergic patients that are hidden under normal analysis conditions.

Most generally protein allergens are identified from raw materials that cause allergic reactions in humans. Examples of raw materials are plant pollens, dust, tissue extracts, fungi, fruit pulp, material containing microorganisms, animal epithelia, insect venoms, just to mention a few. If an allergic reaction is identified, the raw sample is fractionated in order to try isolating and characterizing the antigen responsible for the disease. Nevertheless there are allergenic proteins that are present in very low traces, thus precluding a proper identification by regular means (essentially chromatographic fractionation).

So far only major allergens have been identified: those that have the most important occurrence and that are relatively abundant in the raw materials. There is therefore a lot to be done considering that the allergic disease is increasingly observed in the last decades and therefore it focuses a lot of interest especially in the developing countries. The number of sensitive individuals increases continuously as well as the newly identified allergens, as attested by a rapidly growing number of published papers.

Allergens interact with IgE antibodies (or even possibly IgG) with very specific biochemical recognition of peptide epitopes; however, no specific structures have been identified as being allergenic even if it is demonstrated that an allergic epitope loses its allergy induction (suppression of IgE binding) by just replacing or removing an amino acid within the sequence [14]. This being said it is common to use IgE binding properties to identify novel allergens from crude extracts [15]. It is nevertheless  known  that  immunoglobulins  E  can  also  recognize non-allergenic structures without related allergy symptoms [16].

The binding of a protein allergen to the corresponding IgE occurs between the active recognition site of the antibody and either a linear epitope of one or two dozens of amino acids or a conformational epitope of the antigen. In the first case the same IgE may cross-react with other antigens that share the same epitope, whereas this does not happen with conformational structure docking. This is why patients sensitized against a given allergen family (same epitope) may react to allergens from different species [17, 18].

In this review we highlight the specific phenomenon of amplification of rare proteins in crude extracts in view of detecting novel allergens by means of CPLLs and exemplify several cases of newly discovered allergens of animal and of plant origin.

Current approaches for allergen identification and characterization from raw materials

When dealing with protein allergens it is important to make a distinction between the detection methods and the isolation method that allows perfecting the molecular characterization.

1. The detection of protein allergens

An important step is the detection of allergy against proteins of a given source. The most popular one is based on the interaction with antibodies or immunochemical methods producing either qualitative or quantitative data. Quantitative tests involving enzyme-labeled or radio-labeled IgE antibodies are used for the detection of allergenic activity in a predefined preparation that is chemically attached on a solid support [19,20]. Direct or indirect ELISA-based assays are used to quantify the IgE binding activity against possible sources of allergens [21] or to quantify the presence of allergens in typical extracts [22]. These approaches are used for bulk preparations of allergen sources, but for better detecting the allergenic components of a preparation it is possible to make the immunodetection of allergens after electrophoretic separation of components. The so-called immunoblotting technique [23] is largely used in vitro for the detection of allergens. Briefly this is operated by first making an electrophoretic migration of the protein components of the allergen preparation and then transferring them to a nitrocellulose (or equivalent) membrane. The membrane is then incubated with IgE from the serum of sensitized patient followed by incubation with IgG anti IgE for a classical immunoenzymatic detection. This has been used for the detection of a number of proteinaceous allergens from seeds [24], pollen [25,26] or fruits [27]. Immunoblotting has also been extended to two-dimensional electrophoresis according to similar procedures applied to two-dimensional maps [28]. The separation of component being significantly enhanced, the number of proteinaceous allergens that can be found is larger and is materialized by a spot.

A word is also deserved on several analytical immunoprecipitation methods in a gel such as immunoelectrophoresis [29], crossed immunoelectrophoresis [30] and rocket immunoelectrophoresis [31,32].

2. Isolation methods for allergen characterization

The full characterization of a protein allergen includes not only the establishment of the structure and the physicochemical properties, but also its biological function along with the determination of the epitope structure responsible for the association with a specific IgE. A scheme of full identification and characterization is reported in a review by Mari [33] where most of the steps are listed. To accomplish these tasks it is necessary to purify the allergen by preparative means. In addition to semi-preparative methods such as electrophoresis and capillary electrophoresis, liquid chromatography certainly plays here a critical role with all the possible variants (ion exchange, hydrophobic and affinity chromatography). These techniques can be operated at classical low-pressure or by using specific conditions called high performance liquid chromatography (HPLC). The latter is very effective in separating components from a crude mixture; however, it delivers smaller amounts of pure material as compared to the classical low pressure set-up. The type of chromatographic separation depends on the properties of the allergen. For example protein allergens with an acidic isoelectric point are separated by anion exchange chromatography; hydrophobic allergens are separated using hydrophobic interaction chromatography [34]. These allergens can be glycosylated or not, thus driving the possible affinity separation methods. There are cases, e.g. olive pollen allergens, where the targeted allergen is a glycoprotein (affinity for grafted Concanavalin A [35]), in other cases allergens can be separated using grafted specific antibodies [36], or by affinity chromatography on grafted poly-L-proline [37]; others can be captured by a hydrophobic sorbent [38], therefore via three af-finity chromatographic methods that can be used separately or under a complementary manner.

Most of the time full purification processes imply different chromatographic fractionations including sequences of two or three affinity chromatography columns with complementary properties. Many examples of purification/isolation of allergens of different sources combining different types of liquid chromatography have been reported. Grass pollen allergen isolation, for instance, started in the early ’70s with quite trivial methods of separations. Then chromatographic combinations were used. In an instance a combination of gel filtration, ion-exchange chromatography and preparative isoelectric focusing has been used successfully for the isolation and characterization of a glycosylated protein allergen with a molecular mass of about 10 kDa along with two additional compounds with close acidic isoelectric points [39]. The combination of protein precipitation with ammonium sulphate precipitation followed by ion-exchange chromatography, gel filtration and chromatofocusing allowed also to successfully isolate allergens from grass pollen with great intradermal skin test allergenicity in patients and strong IgE-binding reactivity determined in vitro [40]. Hydrophobic interaction chromatography combined with exclusion chromatography was also used for the separation of grass allergens [41] as well as reverse phase liquid chromatography, due to some hydrophobic character of these proteins [42]. This technique appears useful for the separation into two subgroups of group 5 allergens. Concerning the separation of group 1 grass pollen glycoallergens a combination of ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography appears well adapted for yielding antigens with high IgE reactivity and that are highly frequent in serum from grass pollen allergic patients [43].

Many other examples of protein allergen purification by different chromatographic methods are available with practically all possible variants. This includes dye chromatography for the separation of an allergenic fungal peptide, hypogin [44]; immobilized metal affinity chromatography adopted for the purification of a recombinant protein from dust house mite showing capability to combine with IgE [45]; mixed mode chromatography applied to the separation of birch pollen allergens [46]; lectin chromatography for the separation of a glycoprotein allergen from Aspergillus fumigatus [47] and chromatofocusing in relation to the isolation of allergen Dpt 12, from the house dust mite Dermatophagoides pteronyssinus [48], just to mention a few. These fractionation methods taken individually are quite easy to operate; however, when combinations of them are necessary to reach the purification goal, a number of difficulties have to be overcome.

Both detection and characterization of protein allergens become hard  when  the  targeted molecule is present in trace amounts that could be below the detectability of current analytical methods, thus requiring complex purification processes. It is within this context that it is useful not only to start from a relatively large initial allergenic crude sample, but also to either eliminate the high abundance species that mask the presence of allergen traces or to reduce the dynamic protein concentration range, thus enhancing all protein traces while reducing drastically the major proteins.

The CPLL-based technology for low-abundance protein discovery

It is important to recall here that proteomic analyses are complicated by the presence of high abundance proteins that hide most if not all the very dilute species. To resolve this question the first proposal was their removal, thus rendering the resulting protein mixture better resolvable. Pseudo affinity and affinity columns have been firstly used but quite rapidly immunosorbents, involving specific antibodies against several dominating proteins (e.g. albumin, immunoglobulins, transferrin, alpha-1-antitrypsin and few others, when the sample to analyze is serum), were adopted. In spite of the great technical advance, the process suffers from serious drawbacks such as the removal of many other proteins by non specific binding and the fact that the situation of low abundance proteins is aggravated by further dilution. The benefits of this technology are thus very limited and again they have been challenged [3,49]. In addition the immunodepletion technology applies only to a single type of sample, namely blood serum (or plasma), and is species specific.

It is within this context that the CPLL technology, also frequently classified as “enrichment”, has been proposed and developed.  Actually this methodology not only enriches indiscriminately all low abundance proteins, but importantly enough it reduces the concentration of high abundance species, thus producing a very good resolution of all protein content of a biological sample. CPLL is a collection of millions of hexapeptide ligands each of them grafted covalently on beads that are then mixed together. Each bead comprises million of copies of the same peptide conferring to the solid phase a great binding capacity for protein capturing from any sample. All the beads are mixed together; they act necessarily under the same physicochemical conditions of pH and ionic strength at the same time. This peculiar mode of making affinity chromatography is extensively described and interaction phenomena detailed in a recent book where also many applications are reported along with various recipes for the technology implementation to different types of samples [4].
From the first seminal paper by Thulasiraman et al. [50] a great number of procedures have been reported without changing the principle. Briefly this is a mixed bed of chromatography beads each of them carrying a different hexapeptide affinity ligand (more than a dozen of millions of different peptide com
binations) that is loaded with a large excess of a biological protein extract. Due to the large number of ligands most if not all proteins are targeted.

Under  these  conditions  very  dilute  species  are  progressively concentrated on their respective affinity beads while high-abundance proteins rapidly saturate their affinity ligand. Upon the elimination of protein excess by a simple buffer washing, the captured proteins can be easily harvested. Some proteins are harvested by the same peptide but, by the effect of different affinity constants, an intensive competition takes place  with  protein  displacements  effects  not  correlated  to their relative concentrations. At the same time single hexapeptide ligands display the capability to capture the same protein under different affinity constants completing thus the complex capturing mechanism. On the molecular level the adsorption of proteins by the peptide library is the result of the equilibrium of synergistic and contradictory forces, such as electrostatic attraction or repulsion, hydrophobic associations and hydrogen bonding as detailed in [4].

The protein recovery by elution needs thus to target simultaneously all these interactions otherwise the collection of proteins would only be partial.

This technology is modulated by a relatively large spectrum of physicochemical parameters such as the extent of overloading, the ionic strength, the pH, the temperature, the protein concentration and the presence of competitors. To enhance the concentration of very low abundance species a large sample volume is required, which is an advantage over competitive methods since with very small biological samples (e.g. 10-50µL serum), there would be no chance of finding traces of biologically active proteins. For the protein elution very stringent solutions are necessary because some species are very tightly captured otherwise they would be lost, thus escaping detection. Generally while weakening certain interaction forces others can easily be exacerbated and the protein elution might not be exhaustive. To resolve this question several proposals have been made [51]; one is the use of a solution of sodium dodecyl sulphate (SDS), another one is the direct trypsination of the beads. The first approach applies to all analytical current top-down methods (after elimination of SDS except SDS-PAGE) such as two-dimensional electrophoresis and MALDI. The second one applies to mass spectrometry after peptide fractionation with either HPLC or IEF.  Detailed methodologies can be found in [4].

What is important to keep in mind is that the enrichment operation is directly dependent on a large bead overloading allowing the great concentration of dilute species and the dilution of the abundant proteins when present (Figure 1). The loading increase drives the saturation of beads with rare species with a progressive displacement effect of other species. Below the saturation level (about 10 mg/mL of beads) the compression of the dynamic range is generally low. Under overloading conditions the discovery of novel species becomes very relevant, the total number of species that can be detected could be higher than 3000 as already published for urinary proteins [52] and red blood cell lysates [6]. In an example of proteins from chicken pectoral muscles Rivers et al. [53] observed a progressive discovery of new gene products as the sample loading increased from 20, 50, 100, 250, 500 and 1000 mg of proteins for the same volume of beads (100 µL). The formation of novel high-abundance species when the amount of exposed proteins to the beads was extremely large was also observed.

J J Aller Immuno 2 2 015 Fig1

Figure 1.
Examples of protein enrichment using CPLL.
A: SDS-PAGE of rat serum treated with CPLLs (lane 2) and compar to the non-treated sample (lane 1). Major proteins are considera diminished  and  low-abundance  proteins  undetectable  before  the treatment emerged.
B: 2-DE of human red blood cell lysate treated with CPLL (plate 2)and compared to the non treated extract (plate 1). The major protein hemoglobin (at the right bottom of the plate 1) was largely diluted while many other undetectable proteins came to detection upon the treatment.

Within the domain of proteomics of allergens, also known as allergomics (a bad neologism!), there are two situations to distinguish: (i) the discovery of novel low abundance protein allergens and (ii) the detection and assay of a very dilute given allergen in the original protein extract or in complex food preparations. This distinction is useful for technical approaches that are not exactly the same.
Both use a common process up to the protein enrichment on CPLL beads; then they follow different methodological routes. The discovery of novel allergens is almost a blind process that starts with the CPLL-treated protein extract collected after exhaustive elution from the beads. This mixture is subject to an electrokinetic separation such as SDS-PAGE, 2-DE or IEF and then immunoblotted against the serum of allergic patients. IgEs are revealed by means of a labelled IgG against the human IgE. This results into the localization of bands (SDS-PAGE and IEF) or spots (2-DE) that are topographically localized on the original gel having been used for the immunoblot. At the gel level of the positive signal the gel is sliced or spotted; the collected pieces of gel are ground and proteins are extracted for trypsination and identification by means of sensitive LC-MS/ MS. The identification of allergens is operated with the use of general data banks correlating the peptide composition and its origin (protein name and organism species).  Figure 2 schematically illustrates the entire process.

 J J Aller Immuno 2 1 015 Fig2

Figure 2.
Schematic representation of a discovery route for new allergens using CPLLs. The major key success factor is the total desorption of all cap- tured proteins from the beads.

The detection and assay of a given tracked allergen follow a little different route. The CPLL beads carrying the captured and enriched protein species are directly subjected to trypsination [54, 55] (this can also be done after an exhaustive protein elution) and the collected peptides subjected to an analysis by LC-MS/MS or MRM (multiple reaction monitoring) for quantitation (Figure 3).

J J Aller Immuno 2 1 015 Fig3

Figure 3. Schematic representation of an allergen assay route using the CPLL technology to enhance the signal of low-abundance allergens especially when they are in the presence of complex matrix. Instead of desorbing the captured proteins from CPLL beads, the digestion can easily performed directly on-beads ensuring a complete view of all captured proteins

Low-abundance allergens discovered by means of CPLL

More than 350 plant proteinaceous allergens are officially known as listed by the IUIS Allergen Nomenclature Sub-Committee (http://www.allergen.org/). They come from different sources and there are many variants in their composition. The body entrance routes of contact with the allergenic protein could effectively be of various nature: inhalation (with pollen), contact with skin, adsorption as food. In addition, independently from their body entry point, the allergic symptoms in humans can be widespread: strong or weak, general or localized, transient (more or less rapidly) or permanent. While the majority of known allergens can quite easily be detected, there are others that are very dilute and difficult to find with current methods. Therefore their number is by definition unknown because all those that are below the detection limit are not yet discovered. It is within this context that methods capable to enrich proteins of low abundance, developed in proteomics investigations, can easily be adapted for novel allergen discovery. One very promising enrichment method, as presented in the introduction, is the one involving the use of CPLLs. To date more than five hundred published papers attest the performance of CPLLs in low-abundance proteins enhancement with a strong contribution to the elucidation of proteomes of animal and plant origin with respect to undetectable species. Interestingly, protein markers from a number of diseases have been found and particularly those denominated “early-stage” [9, 11,13] that are at the initial process of a metabolic disorder, thus allowing for more efficient precocious treatments. Since CPLL is widely applicable without restriction of species and type of proteins, it applies indiscriminately to protein allergens.

1. Plant allergens

One of the first papers published on the matter and involving CPLLs dealt with the extension of proteome knowledge and detection of allergens in Hevea brasiliensis latex [56]. In spite of difficulties to manipulate this quite viscous material illuminating results have been achieved. From the initial latex the rubber has been first separated by centrifugation, then the proteins from the clear serum solution have been precipitated by 90% saturation ammonium sulfate and finally treated with CPLLs under physiological conditions.

The native untreated protein sample revealed 234 gene products while the use of peptide libraries added 67 additional new low-abundance gene products. When exposed to human serum from 20 latex-allergic patients a quite large number of electrophoretic protein bands reacted with IgE demonstrating the presence of sensitizing proteins. The patterns as illustrated in Figure 4 are dissimilar from one patient to another indicating that the allergic reaction addresses more than just the previously registered antigens (from Hev b 1 to Hev b 13). Af-ter identification of all the IgE-reactive antigens the following allergenic proteins have been found: Heat shock protein (80 kDa), a proteasome subunit (30 kDa), a protease inhibitor (7.6 kDa) and Glyceraldehyde-3-phosphate dehydrogenase (37 kDa) never described before as allergens.

Another important investigation has been made on the allergens present in cypress (Cupressus sempervirens) pollen [57]. This has been a crucial step in the identification of novel allergens because it represents a widespread and highly invasive allergenic source worldwide. Here the protein extraction was made in the presence of a small amount of a non ionic detergent and 3M urea to improve the solubilization of proteins in phos- phate buffer. Pigments and polymeric material were also eliminated by precipitating all proteins in the presence of ammonium sulfate at 90% saturation. The resulting protein solution was exposed to CPLLs and the collected proteins were compared to the native protein extract. The protein solutions have been analyzed by SDS-PAGE (polyacrylamide gel electrophoresis), two-dimensional electrophoresis and mass spectrometry. A total of 108 unique gene products have been found: 40 from the control sample and 68 supplementary species upon CPLL treatment. Two-dimensional electrophoresis gel plates have been blotted and then exposed to several sera from allergic patients. Twelve IgE-binding proteins were characterized, 9 were already reported as allergens in various sources including the two major known allergens of Cupressaceae (groups 1 and 2). Three IgE-binding proteins, not previously reported as allergens, were newly described: Rab-like protein, involved in membrane fusion, a chaperone protein HSP104 and a Sigma factor sigb regulation protein which is a hydrolase involved in stress regulation mechanism. The known allergens were discovered and characterized by current fractionation methods, whereas the novel gene products were evidenced thanks to CPLL amplification. Seven other IgE-binding antigens with putative allergic effect have also been identified, however, they were already known from other plants. They are Cu–Zn superoxide dismutase, an allergen from olive pollen [58,59]; cytochrome c, corresponding to the group 10 of grass pollen allergens [60]; glucanase-like protein, described as an allergen in olive pollen [61]; lactoylglutathione lyase, also called glyoxalase I, reported as an allergen in rice seeds [62]; malate dehydrogenase, found in ash pollen [63]; phenylcoumaran reductase, described as an allergen in some fruits; and the triosephosphate isomerase protein, present also in oriental Plane tree pollen.

 J J Aller Immuno 2 1 015 Fig4

Figure 4. 1D SDS-PAGE immunoblots of an Hevea brasiliensis latex extract.
Three types of samples were analyzed: untreated (left), treated with Library-1 (center) and treated with library-2 (right) of each group of three strips. The immunoblot was obtained by exposing the samples to the serum of 18 allergic Hevea patients (numbers 1-18 at the top of the figure). Results were compared to two non-atopic donors (numbers 19 and 20 at the top of the figure).  Reproduced from D’Amato et al. [56] with kind permission.

Tropical fruits, such as banana and avocado, are also a large source of allergens. The discovery and identification of allergens in these vegetable tissues is particularly hard because of the poor protein content (banana) and a large amount of lipids (avocado). Banana is a good and energetic aliment for people living in tropical regions. It contains cell antiproliferative activities and antioxidant properties reducing the risks for certain diseases, cancer and cardiovascular issues. This fruit comprises about 20% carbohydrates and as little as 1% proteins. This is not only particularly low, but it is aggravated by the fact that allergens possibly present represent only a small fraction of it. By using CPLLs, it was possible to identify, for the first time, up to 1131 proteins [64]. Among them severalwell-known allergens such as musa a 1, pectinesterase, superoxide dismutase have been detected. Interestingly with CPLLs additional allergens were identified for the first time in banana pulp: lipid transfer protein (LTP) [65], thaumatin-like protein [66], class I chitinase [67], profilin [68], and β-1,3-glucanase [65], respectively. It is to be noted that both class I chitinase [69] and β-1,3-glucanase  seem responsible of the “latex-fruit syndrome” [70] while profilin is involved in the cross-sensitization between pollen and plant-derived foods [71].

The avocado, a fruit of the tropical tree Persea americana, has been quite extensively studied for its content in lipids and oil and also for the presence of many small molecules associated with health benefits. However, almost nothing is known on its proteome composition. A study involving the use of CPLLs has  recently  been  performed  [72]  revealing  the  presence of more than 1000 gene products, most of them of very low abundance. Using in parallel the native protein extract and the extract treated with the combinatorial peptide ligand library it was possible to identify 1012 unique proteins, 648 of them detected thanks to CPLL treatment. Among them two putative proteins already described as allergens have been found. They are the allergen Pers a 1, a class I endochitinase with a mass of 35.5 kDa, and profilin, proposed as an avocado allergen [73] and named as Pers a 4. The latter is found in a large number of vegetables and has been established as widely distributed in nature.  For instance its allergic action has been reported in latex (Hebv 8), melon (Cuc m 2) and banana (Mus a 1). Other proteins of allergenic action found thanks to CPLLs are 1,3-beta-glucanase, thaumatin-like protein, involved in host defence processes described as allergens in pollen and fruits [74], and polygalacturonase that has been described as allergenic protein in other species [75].

Various other reports have been published around the detection of novel allergens from plant origin; this is the case of maize kernels [76], olive oil, where the amount of proteins is extremely low due to the “hostile” apolar environment [77] and also in some non-alcoholic beverages that are supposed to contain almond extracts [78].

2. Allergens of animal origin

The sources of animal allergens are also very numerous. They can be from common animals such as mammals (cat, horse, etc), from fish and sea products (e.g. salmon, yellowfish tuna), from animal products (e.g. milk and eggs), from small marine organisms (e.g. arthropods and molluscs) and from microorganisms (e.g. fungi). A large list is given by the IUIS Allergen Nomenclature Sub-Committee (http://www.allergen.org/).

Within the protein allergens of animal origin are also very low-abundance species that are normally undetectable if not “amplified”. The situation is even further aggravated compared to plant extracts by the fact that most of the time the dynamic range in animal extracts is extremely large, encompassing up to more than 10 orders of magnitude. Therefore the amplification of dilute proteins is unfortunately not enough to bring these species at the level of detection because abundant proteins largely mask the analytical signals. In this situation, the CPLL technology is well adapted to reduce the dynamic range, as repeatedly reported. Within the domain of allergens of animal origin the following examples are representative of many different situations: discovery and/or detection of allergens in cattle milk, in insect venom and in eggs.

Milk is a common food for young mammals and a source of proteins and calcium for adult humans. It contains more than 3% proteins, among them 80% caseins, 20% albumin, lactoglobulin and then a large number of other proteins at low or very low concentration. One of the most relevant proteomics investigations of bovine milk whey involving the use of CPLL has been published a few years ago [79] with the discovery of few hundred gene products, 100 of them never disclosed before. In spite of its large use in human diet, milk and milk derivatives are not exempt from peculiar drawbacks such as intolerance that in certain cases is attributed to the presence of protein allergens. The well-known milk allergens are caseins, lactalbumin, lactoglobulin and, to minor extent, lactoferrin. The analysis of milk whey after treatment with CPLL revealed additional interesting data. Technically the milk whey was first obtained from fresh milk by ultracentrifugation and, after addition of antiproteases, it was treated with CPLLs. After washing, the desorbed proteins were analyzed by isoelectric focusing, then immunoblots were made against the blood serum of 25 milk-allergic patients. All the process was also confronted with a general control (whey milk non-treated with CPLLs). The reaction with IgE from patient’s serum evidenced a number of bands of putative known allergens and several bands of low-abundance allergens undetectable in the control IEF strip. Although the immunoblot of the control did not show any presence of immunoglobulin reaction, in the eluate of CPLLs it was possible to clearly find a series of small bands attributable to polymorphic immunoglobulins (see Figure 5).  Beyond these data many other minor nonidentified IgE positive protein bands were detected from most patients in the acidic side of the IEF (pI between 3 and 4) as well as lactoferrin detectable at a pI around 9.5.

Upon treatment with CPLLs and subsequent analyses with proteomic techniques, Coscia et al. [80] investigated human colostrums where from they detected, in addition to many novel interesting milk proteins, galectin-7 and amyloid P-component (both probably involved in the regulation of the normal cell growth) and α-S1-casein that can be considered as allergens and that can be the cause of certain infant sensitization to cow’s milk.

Recently Siciliano et al. [81], while investigating the thermal treatment  consequences  of  milk  and  derivatives,  reported the detection of a lactose glycation reaction happening at the ε-amino group of lysine residues increasing the potential risk of allergen reactivity in infants by these novel hapten-like antigens. They strongly supported the idea of mandatory analytical proteomics methodologies based on mass spectrometry after the amplification of products derivatives with CPLLs.

 J J Aller Immuno 2 1 015 Fig5

Figure 5.
Immunoblotting from an IEF analysis of milk whey proteins (native “A” and after CPLL treatment “B” using two libraries “Lib-1” and “Lib-2”) against sera samples from allergic patients.
A: The “wp” lane is the crude whey protein mixture stained with Coomassie blue. The following lanes from left to right are immunoblots of the same protein mixture with different serum samples (from “e” to “p”). “ct” is the negative control.
B: Immunoblotting from an IEF analysis of proteins desorbed from peptide libraries. “Lib-1” and “Lib-2” are Coomassie stained eluates.

All others are as described for panel A, all eluted proteins being from Library-2, and “ct” being the negative control. For both panels A and B, regions indicated by “cas”, “α + β”, “Ig” and “Lf ” mean, respectively, casein, α-lactalbumin and β-lactoglobulin, immunoglobulins and lactoferrin. Reproduced from D’Amato et al. [79] with kind permission.

Hen’s eggs have quite extensively been explored by using proteomics investigations after CPLL treatment and focusing respectively on egg white [82] and egg yolk [83]. The former paper allowed detecting a much larger number of gene products over what was known at the date, with 147 novel unique proteins. The latter also increased the number of known gene products from what was known to 255 novel unique proteins. They comprise a very complex mixture of proteins among them the best known being ovalbumin, ovotransferrin, ovomucoid and lysozyme.

Eggs are largely used as food because of their nutrition value and in the food industry for their foaming, gelling and emulsifying properties. Eggs were and still are also used for the preparation of some vaccines that generate some adverse effects that are attributed to the presence of traces of ovalbumin, a well-known potential allergen. Actually eggs represent the second most frequent source of allergic reactions in children; this reaction is related to the resistance of egg proteins against digestion, which allows the direct absorption at the level of the intestinal mucosa tract. The effects of eggs in the food matrix and processing have been recently described [84]. Other protein traces with potentially larger immunological reactivity may also be present contributing to allergic sensitization and unpleasant effects. It is within this context that Martos et al. [85] investigated the discovery of novel allergens in egg white by using the CPLL technology. Beside the well-known allergens they found two additional minor egg white proteins, identified as ovoinhibitor and clusterin, both reacting with serum IgE from egg-allergic patients. Additionally it was reported that the immunoreactivity of previously digested egg white proteins against IgE was not affected by the presence of egg yolk. In another paper the same authors [86] discussed the characterization of white, yolk, and shell proteins using modern proteomics investigations in relation to egg allergens native or after processing.

Honeybee venom is another source of allergic reaction. It comprises a large number of molecules and polypeptidic components. This list was limited to only 30 till recently, when it has been largely extended by the use of CPLLs [87]. Among the protein components are defense toxins that may also exert a role in social immunity. With the use of combinatorial peptide ligand libraries 102 unexpected proteins have been identified. Twelve protein allergens have been found and reported to occur in 0.8-5% of general population going from just sensitization to life threatening. The list of novel proteins may also comprise additional potential venom allergens that are not yet functionally characterized. In a similar study Matysiak et al.[88] explored the honeybee venom proteome after enrichment strategies involving CPLLs and solid-phase extraction followed by shotgun proteomics analysis,, thus yielding a significantly increased number of identified peptides in these samples, compared to the existing literature. Here also in addition to the known and reported allergens the authors found four additional new uncharacterized allergic proteins.

To complete this section it should be mentioned that attempts have been made to detect fungal allergens in invasive aspergillosis after the use of CPLLs for low-abundance protein enhancement [89] a major life threat with diagnostic difficulties. The study had been operated by the CPLL treatment of both serum and bronchoalveolar lavage followed by DIGE analysis from a mouse model of invasive pulmonary aspergillosis. This approach resulted in the identification of fungal protein corresponding to the major allergen Asp f 2. These data along with current microbiology and inflammatory host response provide possible insights to the physiopathology of this invasive contamination.

To complete this section it should be mentioned that attempts have been made to detect fungal allergens in invasive aspergillosis after the use of CPLLs for low-abundance protein enhancement [89] a major life threat with diagnostic difficulties. The study had been operated by the CPLL treatment of both serum and bronchoalveolar lavage followed by DIGE analysis from a mouse model of invasive pulmonary aspergillosis. This approach resulted in the identification of fungal protein corresponding to the major allergen Asp f 2. These data along with current microbiology and inflammatory host response provide possible insights to the physiopathology of this invasive contamination.

3. Allergens in food

Allergies from food are gradually increasing over the years in all countries as exemplified with children suffering from these ailments attested in various reviews [90]. Therefore proteins are not all good for eating at least related to the induction of allergic reactions that can have dramatic effects and can go up to life threatening. Actually in food proteolysis-stable proteins are present; they can induce a sensitization at the level of intestinal tract where the mechanisms starts [91]. In spite of the fact that a great knowledge has been developed around allergic components that can be present in food, a lot remains to do to prevent these reactions due to the fact that food composition is not often well and exhaustively documented.

The detection of protein allergens in food products is currently performed via ELISA-based methods and PCR procedures; however, this is applicable to proteins that are targeted in advance. When searching for potential allergens from ingredients that are not listed as composing the food or allergens that are below the sensitivity level of current methods, this task is unachievable. Aggravating the situation is the fact that possible present allergens are hidden by the large amounts of food matrices that mask and prevent their detection and therefore yield negative results or even false positive ones [92].

Various types of food comprise proteinaceous allergens of animal and vegetal sources such as fish, shellfish, eggs, milk, wheat flour [93], soybeans [94], seeds, nuts and fruits. Due to the widespread presence in food preparations peanuts focus the attention due to the seriousness of the biological consequences of allergens present therein. That is why a number of investigations have been made to detect peanut traces in industrial food such as ice cream [95] and chocolate products [96]. It is also speculated that a source of allergens could be related to transgenic organisms transformed and/or used as food additives [97]. The modification of a genome could alter even a little protein expression with respect to allergenic components and it is very complex to detect novel allergens that could be present at very low traces. To monitor transgenic material in food a real-time immuno-PCR assay has been described, providing detection of very low traces of transgenic proteins (100 pg/mL LOD) [98-100]. Even if this is a good indication, it does not exhaustively provide data about the possible allergens present. Conversely, but within the same context, transgenic organisms can be generated with the objective to reduce the content of allergenic proteins. This is the case of rice seeds known to comprise major potential allergens [101]. In certain transgenic of hypo-allergenic rice lines, the content of allergens is largely diminished almost annihilating the IgE binding in the majority of sensitive patients.

In this emerging domain CPLLs could play a key role with their capability of enhancing very low abundance allergens bringing them to the level of detectability and using current proteomics methodologies. This will indicate not only the presence of all the allergens against a given patient but it can also tell where the allergen comes from (species and type of organ). Technically a food extract is exposed to the serum of the patient and, by using technologies described in a previous section, it is possible to formally identify the name of the allergens. The diagnosis for individuals is thus possible; however, what is very challenging is the general detection of allergens even at trace levels since this type of analysis would address only known allergenic proteins unless CPLL is applied for allergen traces enhancement.

A  concrete  example  is  given  by  peanut  allergen  presence in food. The occurrence of this type of allergy is so frequent and serious that traces of peanuts are tracked in various food preparations. This has recently been searched in baked cookies with the help of CPLLs as amplification system to track minor traces of peanut allergens [102]. Since even cookies are quite complex compositions, the authors insisted on the fact that very sensitive methods as well as very rigorous approaches are needed to detect traces of allergens since no generic platforms are applicable and also because the published procedures lack details for a proper detection workflow. Basically 1-5 grams of backed cookies were ground to a particle size below 250 μm and extracted with Tris-buffered saline. The protein extract was then treated with CPLLs according to current protocols [4] under very large overloading. After the collection of captured proteins, followed by trypsination, the peptides were analyzed by LC-MS/MS with protein identification. The applied workflow here was not designed to discover novel allergens but rather to detect very small traces of peanut allergens in food contamination. This is very challenging because the tracked protein allergen traces are obscured by the food matrix. Under normal conditions (no enrichment) the authors underline the impossibility to detect peanut allergens in cookies and only by selecting the appropriate allergen and using th CPLL technology they reached the detectability level. Peanut allergens nicely enriched with CPLLs were Ara h 1 and Ara h 3 (Figure 6); however, in the presence of a food matrix the best enrichment occurred with Ara h 3 with about 10x enhancement. Peptides representing the allergen evidenced by mass spectrometry and quantified by selective reaction monitoring (SRM) confirmed that it was possible to detect peanut presence at trace levels of about 10 µg per gram of food matrix.

Beverages are also a potential source of allergies when they contain extracts of animal or vegetal sources. In a study around the contamination of white wines by proteins that are used to clarify from sediments due to some flocculation phenomena [103] it has been found that traces of casein were still present. Actually casein is added to eliminate wine precipitates and its presence is reduced by the fact of its poor solubilization at wine pH and by addition of bentonite. In spite of these treatments, traces of casein are still present. This has been demonstrated by using CPLLs with a detection limit estimate of 1 µg per liter of wine. This is an important finding because casein is known as a major food allergen in bovine milk. This risk adds to chitinase and lipid transfer protein (LTP), that are well-known allergens that could come from grape berries.

J J Aller Immuno 2 1 015 Fig6Figure 6. Enrichment of major peanut allergens through combinatorial peptide ligand libraries. Reproduced from Pedreschi et al. [102].

Some aperitifs contain extracts from plant origin as for instance Braulio, an Italian 21% alcohol aperitif that is prepared using extracts from more than ten herbs and berries, among which Gentiana alpina, Artemisia absinthium, Achillea moschata and Juniperus communis. These extracts comprise protein traces that are detectable in the final beverage after the treatment with CPLLs. In a recent proteomics study [104] the presence of 29 gene products such as PR5 (parasite resistance protein) and Jun r 3.2, both known as allergens for humans, has been reported.

To complete this summary review it is also recalled here that beer, as fermentation beverage, can contain potential protein allergen traces that can be evidenced upon enrichment with CPLLs [105]. Their origin is to be found from the transformation of ingredients to beer. The identification of hordein-derived peptides from this study raises immunological concerns that probably need to be addressed soon or later.

Conclusions

CPLL is a well established technology in proteomics investigations allowing the discovery of thousands of low-abundance proteins from various sources, but it is still emerging with respect to new allergen discovery. Nevertheless its efficiency has been demonstrated in a good number of cases with results that open the possibility to expand its use towards many rare allergens present in complex food matrices. Especially powerful is the combination of this technology with proteomics approaches involving 2-DE, LC-MS/MS and MRM. The only bottleneck that is under progressive elimination is the availability of complete database of peptides for the identification of proteins, the latter being frequently made by analogy between species. Beyond the discovery aspect, this approach could bring information on IgE binding processes. This would elucidate the reasons why the reaction between allergens and IgE does not always produce allergic reactions. Progress to be made is around the resolution improvements of two-dimensional electrophoresis for isoforms separations and around extreme isoelectric points. Moreover with the extension of performance of CPLLs towards large pH ranges and also towards hydrophobic regions, as already described [5], many novel discoveries are expected.

In addition to what has been reported in this review, limited though to findings mostly related to CPLL treatment of various foodstuff, the EFSA (European Food Safety Authority, Parma, Italy) has recently published a massive volume (286 pages), which is an update of previous opinions of their panels of experts relative to food ingredients or substances with known allergenic potential listed in Annex IIIa of 2003/89/EC [106]. This volume includes cereals containing gluten, milk and dairy products, eggs, nuts, peanuts, soy, fish, crustaceans, molluscs, celery, lupin, sesame, mustard and sulphites. The opinion relates to immunoglobulin (Ig)E- and non-IgE-mediated food allergy, to coeliac disease and to adverse reactions to sulphites in food. It includes information on the prevalence of food allergies in unselected populations, proteins identified as food allergens, cross-reactivities, the effects of food processing on the allergenicity of foods and ingredients as well as methods for the detection of allergens and allergenic foods. This volume is certainly worth consulting.

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Cite this article: Boschetti E. The Discovery of Low-Abundance Allergens by Proteomics Analysis Involving Combinatorial Peptide Ligand Libraries. J J Aller Immuno. 2015, 2(2): 015.

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