Next Article in Journal
Onion (Allium cepa L.)-Derived Nanoparticles Inhibited LPS-Induced Nitrate Production, However, Their Intracellular Incorporation by Endocytosis Was Not Involved in This Effect on RAW264 Cells
Next Article in Special Issue
LC-ESI/LTQ-Orbitrap-MS Based Metabolomics in Evaluation of Bitter Taste of Arbutus unedo Honey
Previous Article in Journal
Stemodane Diterpenes and Diterpenoids: Isolation, Structure Elucidation, Biogenesis, Biosynthesis, Biological Activity, Biotransformations, Metabolites and Derivatives Biological Activity, Rearrangements
Previous Article in Special Issue
The Comparison of Physicochemical Parameters, Antioxidant Activity and Proteins for the Raw Local Polish Honeys and Imported Honey Blends
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 26
Issue 9
10.3390/molecules26092762
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Mining the Royal Jelly Proteins: Combinatorial Hexapeptide Ligand Library Significantly Improves the MS-Based Proteomic Identification in Complex Biological Samples

by
Eliza Matuszewska
1,
Joanna Matysiak
2,
Grzegorz Rosiński
3,
Elżbieta Kędzia
4,
Weronika Ząbek
5,
Jarosław Zawadziński
6 and
Jan Matysiak
1,*
1
Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6 Street, 60-780 Poznań, Poland
2
Faculty of Health Sciences, Calisia University, Kaszubska 13 Street, 62-800 Kalisz, Poland
3
Department of Animal Physiology and Development, Faculty of Biology, Adam Mickiewicz University in Poznan, Uniwersytetu Poznańskiego 6 Street, 61-614 Poznań, Poland
4
Department of Innovative Biomaterials and Nanotechnologies, Institute of Natural Fibres and Medicinal Plants—National Research Institute, Wojska Polskiego 71B Street, 60-630 Poznań, Poland
5
Department of Pediatric Gastroenterology and Metabolic Diseases, Poznan University of Medical Sciences, Szpitalna 27/33 Street, 60-572 Poznań, Poland
6
Department of Physiotherapy, Poznan University of Medical Sciences, 28 Czerwca 1956 r. 135/147 Street, 61-545 Poznań, Poland
*
Author to whom correspondence should be addressed.
Molecules 2021, 26(9), 2762; https://doi.org/10.3390/molecules26092762
Submission received: 15 March 2021 / Revised: 30 April 2021 / Accepted: 4 May 2021 / Published: 7 May 2021
(This article belongs to the Special Issue Progress in Analytical Methods for the Characterization, Quality and Safety of the Beehive Products)
Browse Figures
Versions Notes

Abstract

:
Royal jelly (RJ) is a complex, creamy secretion produced by the glands of worker bees. Due to its health-promoting properties, it is used by humans as a dietary supplement. However, RJ compounds are not fully characterized yet. Hence, in this research, we aimed to broaden the knowledge of the proteomic composition of fresh RJ. Water extracts of the samples were pre-treated using combinatorial hexapeptide ligand libraries (ProteoMinerTM kit), trypsin-digested, and analyzed by a nanoLC-MALDI-TOF/TOF MS system. To check the ProteoMinerTM performance in the MS-based protein identification, we also examined RJ extracts that were not prepared with the ProteoMinerTM kit. We identified a total of 86 proteins taxonomically classified to Apis spp. (bees). Among them, 74 proteins were detected in RJ extracts pre-treated with ProteoMinerTM kit, and only 50 proteins were found in extracts non-enriched with this technique. Ten of the identified features were hypothetical proteins whose existence has been predicted, but any experimental evidence proves their in vivo expression. Additionally, we detected four uncharacterized proteins of unknown functions. The results of this research indicate that the ProteoMinerTM strategy improves proteomic identification in complex biological samples. Broadening the knowledge of RJ composition may contribute to the development of standards and regulations, enhancing the quality of RJ, and consequently, the safety of its supplementation.

1. Introduction

Bee products are unique, complex mixtures of biologically active compounds produced by honeybees (Apis mellifera). They exhibit strong health-promoting properties, appreciated since ancient times and used for medical purposes. Until now, they are recommended as drugs within the branch of alternative medicine called apitherapy [1]. One of the most sterling bee products, along with honey, bee pollen, and propolis, is royal jelly (RJ). It is a semi-liquid, milky white or yellowish secretion of worker bees’ salivary glands. Its sour-bitter-sweet taste (resulting from its acidic pH) and characteristic phenolic smell make it mostly recognizable among the so-called “superfoods” [2]. The term “superfoods” refers to foods that have beneficial effects on human health due to their rich nutrient content [3], and RJ fits this description well.
Chemically, fresh RJ consists of about 60–70% water, 11–23% carbohydrates, 9–18% proteins, 3–8% lipids, and small amounts of vitamins, minerals, free amino acids, and other constituents [2,4,5]. According to the literature reports, RJ is nonhomogeneous, displaying high variability between its main components [5,6,7,8,9,10]. Factors responsible for that variability are the metabolic and physiologic condition of worker bees and larval age [10,11,12], honeybee strain [13], place of origin, and year and season of the collection [11]. Since the fluctuations in RJ composition may significantly influence its biological activity, standardization, and quality control of RJ and other bee products seem to be an essential issue.
In a beehive, RJ serves as superior nourishment for all bee larvae until the prepupal stage. However, the honeybee queen is fed exclusively with RJ for its lifetime [14]. Since only the queen is able to reproduce, the epigenetic influence of RJ in the sexual maturation of the female bee larvae is unquestionable. Although it is not thoroughly investigated yet, how RJ provokes the queen’s development, monomeric major royal jelly protein 1 (MRJP1; royalactin), belonging to a major royal jelly proteins (MRJP) family, has been suggested to influence the larva differentiation into a bee queen [15]. Nevertheless, the mechanism of RJ’s action is probably more complicated, dependent not on the single compound but on the unique molecular blend found exclusively in RJ [16]. Hence, taking into account that RJ is the exceptional food of the bee queen, but it is also used in the treatment and prevention of some diseases, there is an urgent need to characterize the complete RJ composition, including macro- and micro-molecules, along with their biological functions. This will contribute to explaining the effects of royal jelly on the human body and also its beneficial and adverse properties in relation to both humans and animals.
Benefits arising from RJ consumption attract people to include it in their diet. However, the use of not thoroughly tested products poses a risk of side effects, such as inflammatory and allergic reactions, dermatitis, asthma, respiratory stress and bronchospasm, gastrointestinal problems, intestinal bleeding, and even death. Most of the factors responsible for both beneficial and harmful effects caused by RJ intake remain uncharacterized. Hence, taking into account that proteins are mostly accountable for the biological activity of the product, in this research, we aimed to broaden the knowledge of the proteomic composition of fresh RJ.
According to the available literature, several analytical methods have been proposed for the proteomic analysis of RJ. Since MRJPs are easily soluble in water, most of the researchers have extracted proteins contained in RJ using ultrapure water or buffer solutions [17,18,19,20,21,22]. Hence, in this study, we chose water as an optimal solvent. RJ is a complex product eminently rich in proteins [23]. Therefore, in other studies conducted worldwide, before identification, protein extracts were subjected to tryptic digestion and fractionated, using liquid chromatography or isoelectric fractionation approach. The identification analyses were performed mainly by electrophoretic techniques or mass spectrometry [17,18,19,20,21,24,25,26]. In this study, we decided to utilize proteolytic digestion, nanoLC (nano-liquid chromatography) and tandem MALDI-TOF/TOF (matrix-assisted laser desorption/ionization—time of flight/time of flight) mass spectrometer.
In the proteomic investigation of natural products such as RJ, a major challenge is analysis of low-abundant proteins. Because there are significant differences in the concentrations of different proteins contained in RJ with a high content of MRJPs fraction, it is important to focus on proper samples preparation for proteomic analyses. However, according to the literature data, any previous study devised an efficient method for sub-proteome isolation. Therefore, we proposed the ProteoMinerTM (Bio-Rad, Hercules, CA, USA) kit for proteins purifying and concentration. ProteoMinerTM uses combinatorial hexapeptide ligand libraries, and it is a protein enrichment strategy allowing capturing low- and very low-abundance proteins. Such prepared RJ protein extracts were analyzed with advanced nanoLC-MALDI-TOF/TOF MS/MS (nano-liquid chromatography—matrix-assisted laser desorption/ionization—time of flight/time of flight mass spectrometry/mass spectrometry) technique. To the best of our knowledge, according to the available literature, it is the first attempt to analyse the proteomic fraction of RJ samples using ProteoMinerTM protein enrichment approach.

2. Results

The methodology proposed for the analysis of RJ samples allowed us to identify a total of 86 proteins taxonomically classified to Apis spp. (Table 1). Among them, 50 proteins were detected in “crude” RJ water extracts, which were not pre-treated with ProteoMinerTM kit, and 74 proteins were found in extracts enriched with this commercial technique (total in all fractions). The numbers of proteins identified in “crude” extracts, extracts pre-treated with ProteoMinerTM, and both in “crude” and ProteoMinerTM pre-treated extracts are presented in Figure 1.
In total, 35 proteins, accounting for 41% of all proteins identified in this study, belonged to the MRJPs family, responsible for honeybee nutrition and development (Figure 2A). Among them, fragments of MRJP1—MRJP7 and MRJP9 have been detected. The next important functional class of proteins were enzymes (20 proteins; 23% of all the identified proteins), with glucose dehydrogenase, alpha-glucosidase, venom acid phosphatase, lysozyme, and chymotrypsin inhibitor, among others. Into the most abundant protein classes, we also ranked defensins (7 proteins; representing 8% of all identified proteins), and binding proteins (6 proteins, accounting for 7% of all identified proteins), including regucalcin-like proteins. Additionally, four of all 86 proteins (5%) were classified into three other functional groups: remodeling proteins, snRNA processing proteins, and venom carbohydrate-rich proteins.
Moreover, ten identified proteins (11% of all identified proteins) were hypothetical proteins whose existence has been predicted, but there is not any experimental evidence proving their in vivo expression. Additionally, four uncharacterized proteins (5%) of unknown functions have been detected.
Proteins identified separately in extracts not pre-treated with ProteoMinerTM enrichment technology were mainly MRJP (28 proteins out of 50, which represents 56% of proteins identified only in non-pretreated samples). The next functional classes of proteins included enzymes (6 identified proteins; 12%), defensins (3 proteins; 6%), binding proteins (3 proteins; 6%), and others (4 proteins; 8%). In these not-enriched samples, five hypothetical proteins (accounting for 10% of proteins identified in non-enriched samples) and one uncharacterized protein (2%) have been detected (Figure 2B).
Considering only extracts pre-treated with ProteoMinerTM kit, the protein functional class distribution was comparable to overall results obtained from all compiled samples. MRJP (32 fragments) constituted 43% of all proteins identified in ProteoMinerTM fractions. Besides, the most numerous classes were enzymes (17 proteins; 23% of proteins identified only in ProteoMinerTM pre-treated samples), followed by defensins (6 proteins; 8%), binding proteins (6 proteins; 8%) and others (3 proteins; 4%). In those enriched samples, we also identified 6 (8% of all proteins identified in pre-treated fractions) hypothetical proteins and 4 (6%) uncharacterized proteins (Figure 2C).
Application of the ProteoMinerTM technique resulted in expanded identification of proteins that belonged to almost all functional groups. Within the MRJPs group, 9 out of 35 (25.7%) MRJPs identified in total in this study were detected only in ProteoMinerTM fractions. Moreover, 8 out of 19 (42.1%) enzymes identified in total were only detected in pre-treated samples. Regarding defensins, as many as 4 additional proteins belonging to this functional group were identified through the use of ProteoMinerTM, whereas only three defensins were identified in the unpurified samples (meaning that 57.1% of all identified defensins were detected using the ProteoMinerTM technique). Similarly, out of 6 identified in total binding proteins, 3 of them (50%) were detected only in ProteoMinerTM fractions. These results clearly indicate that the ProteoMinerTM strategy significantly improves proteomic identification in complex biological samples, increasing the number of some identified functional proteins even more than twice.
Moreover, compared to “crude” extracts, application of the ProteoMinerTM kit enabled the detection of 5 more hypothetical proteins (out of 10 hypothetical proteins identified in total in this study) and 3 more uncharacterized proteins (out of 4 uncharacterized proteins identified in total in this study). These improvements in identification rate are especially beneficial when trying to find new proteomic features in bio-matrices represented by RJ.

3. Discussion

The goal of this study was to characterize the protein-peptide composition of fresh RJ using advanced nanoLC-MALDI-TOF/TOF MS/MS (nano-liquid chromatography—matrix-assisted laser desorption/ionization—time of flight mass spectrometry/mass spectrometry) technique. Despite its many unquestionable advantages, including versatility, ease of use, cost-effectiveness, and possibility to detect biomolecules in extremely low concentrations (even close to sub-femtomoles), the MALDI-TOF MS technique also has some limitations [27]. One of the most challenging issues is the detection of low-abundance proteins and peptides in very complex biological matrices. The presence of high concentration proteins and also other constituents, like lipids and salts, prevent direct analysis of all molecules contained in the sample [28]. Thus, proteins and peptides in low concentration ranges usually remain undetected. To deal with this problem, before MS analyses, biological samples must be purified, and their complexity and dynamic range need to be reduced. In this study, we applied a novel strategy based on a combinatorial hexapeptide ligand library (ProteoMinerTM). ProteoMinerTM technique, by binding proteins to hexapeptides (through protein affinity interactions), resulted in reducing high-abundance proteins and enriching low-abundance proteins in RJ samples [29,30,31]. In this research, using the ProteoMinerTM technique significantly improved the number of identified proteins, from 50 in non-enriched RJ water extracts to 74 in ProteoMinerTM pre-treated extracts.
Application of the proposed methodology resulted in the identification of a total number of 86 proteins (from all samples and ProteoMinerTM fractions) taxonomically restricted to Apis spp. (Apis mellifera, Apis cerana, Apis dorsata, and Apis florea). As expected on the basis of available literature, the major royal jelly proteins family constituted the largest group. We identified 35 MRJPs or their precursors, which stands for 41% of the number of all identified proteins. Our results reflect previous independent proteomic studies, which reported that MRJPs account for up to 82–90% (w/w) of all proteins found in RJ [32,33,34]. It is also worth noting that in our study of only crude RJ, the percentage of MRJPs in relation to all identified proteins was as high as 56%. These results prove the utility of the ProteoMinerTM enrichment technique in enhancing the protein identification ability by MS approaches.
MRJPs are a family of nine proteins, of which MRJP1, called apalbumin or royalactin, is the most abundant and also the first MRJP to be identified in 1992 [35]. It has been suggested that MRJPs have mainly nutritional and developmental functions. Moreover, MRJP1 has been reported to exhibit antibacterial and antiproliferative activity [14,36]. Interestingly, the effect of MRJPs and whole RJ on honeybee queen development is not only due to the nutritional properties. MRJP1, along with protein named apisimin, by specific complex formation, provide the optimal RJ viscosity. The viscosity is crucial for holding queen larvae on the surface of the comb cells, which are orientated vertically and open downwards [37,38]. The formation of MRJ1-apisimin polymer is strongly pH-dependent. Long structures are formed at pH about 4.0, measured in fresh RJ [37,39]. The specific acidity is sustained by the fatty acids content produced in the bees’ mandibular glands.
The second most-abundant functional group of RJ-identified proteins in this study was enzymes. This group includes some important enzymes crucial for carbohydrate metabolisms, like glucose dehydrogenase, alpha-glucosidase and glucose oxidase. Those enzymes are suggested to play an essential role in facilitating the larvae’s digestion of sugars, which are found in RJ in large quantities [19]. The next relevant enzyme identified in our study was lysozyme isoform 1 and 2. Lysozyme is a protein commonly found in animals. It is a natural component of various secretions such as tears, saliva, mucus and milk. Lysozyme exhibits a strong antimicrobial activity, which in beehive prevents bacterial infection and disease [40,41]. Furthermore, because of their human benefits, the antibacterial activities of RJ are worthy of notice. These properties were reported as far back as 1939 [42]. According to the literature, besides the lysozyme, a 5.5 kDa defensin peptide named royalisin, and 10-hydroxy-2-decenoic acid (10-HDA) are mainly responsible for the antibacterial activity of RJ [43,44,45]. Antimicrobial properties, along with anti-inflammatory, immunomodulatory, antioxidant, moisturising, toning, and anti-ageing activities, make RJ a perfect dermatological product. Hence, RJ is used for treating various skin lesions and wounds [46,47]. Antibacterial defensins, including royalisin preproprotein (named defensin-1 preproprotein), were also identified in this study.
However, among the enzymes included in RJ, we found venom acid phosphatase Acph-1-like and venom serine protease 34. These proteins, along with MRJP8, MRJP9 and icarapin variant 2 precursor (also found in this study), are classified as bee venom allergens (based on Allergen Nomenclature Database, www.allergen.org, accessed on 8 March 2021). Allergens are responsible for the occurrence of allergic reactions in humans and animals after exposition to bee products [48,49,50]. The highest risk of an allergic reaction, both local or systemic, is associated with exposure to Hymenoptera venom, especially after the insect sting. Since allergens are present also in other bee products, like RJ (what was proven in this study), bee products should be consumed with caution and in moderation.
According to literature data, allergy to royal jelly is most common IgE-mediated, but contact allergic reactions (contact dermatitis) are also possible. IgE allergic reactions to royal jelly cause symptoms such as urticaria, angioedema, eczema, allergy rhinitis, conjunctivitis, bronchospasm or even anaphylactic shock [51,52,53]. Patients with bronchial asthma and atopic dermatitis are particularly predisposed to severe reactions to RJ. Even 17% adult patient with asthma [54] and one third of patient with atopic dermatitis [55] have specific IgE to RJ. Reported cases of allergy to royal jelly concern both people who have consumed this product for the first time or after long use. Allergy reaction to RJ after the first use is possible because of cross-reactivity between RJ and other allergens. Cross-reactions between RJ and bee venom can be associated with proteins found in our study—venom acid phosphatase Acph-1-like, venom serine protease 34, MRJP8, MRJP9 and icarapin variant 2 precursor. Moreover, cross-reactions are also possible with very typical allergens like house dust mite (Dermatophagoides farinae, Dermatophagoides pteronyssinus), German cockroach or crab [55]. In conclusion, RJ can cause an allergic reaction because it contains allergenic proteins and, further, because of cross-reactions with typical allergens. Patients with venom allergy, asthma, atopic dermatitis, and allergic rhinitis should be careful before consuming RJ.
Additionally, to minimalize the risk of dangerous adverse effects, bee products should be thoroughly tested for allergens, both qualitatively and quantitatively. However, despite its undeniable beneficial pro-health properties, the use of RJ in people with local and/or systemic Hymenoptera venom allergic reactions should be avoided.
Although in this study we focused mainly on nutritive MRJPs, and enzymes, defensins, and allergens, we also identified other functional groups of proteins included binding proteins, chymotrypsin inhibitors, protein SERAC1-like, and integrator complex subunit 7-like isoform 1. Those proteins show mainly regulatory activities.
The most interesting group of the identified biomolecules, regarding the possibility of learning about new properties and functions of royal jelly, are hypothetical and uncharacterized proteins. In this study, we detected 10 hypothetical proteins whose existence has been predicted based on the genetic information, but there is any experimental evidence proving their in vivo expression. The functions of these proteins, as in the case of uncharacterized proteins, have not been investigated yet. For these proteins, we performed BLAST analysis (https://blast.ncbi.nlm.nih.gov/, accessed on 11 January 2021) to find similar regions in various biological sequences. This approach helps to assess functional relationships between proteins and identify members of protein families. Results of our analysis are presented in Table 2.
For the identified gi|110756431 protein (Predicted: hypothetical protein LOC725074 [Apis mellifera]), we found a significant alignment sequence referred to omega-conotoxin-like protein 1 [Apis mellifera]. This protein was also found in bee venom in our previous studies, based on HPLC, nanoLC-MALDI-TOF/TOF MS/MS, and LC–ESI–QToF methodology [56,57]. With a high degree of probability, we can conclude that the molecule we have identified corresponds entirely to the results obtained from the BLAST platform, as both query coverage and per cent identity equal 100%. It means that our identified sequence exactly corresponds to conotoxin-like protein 1. As far as we know, this protein has never been reported to be included in RJ before. Detection of conotoxin-like protein 1 in royal jelly is a particularly important discovery in terms of the safety of RJ and also its potential therapeutic use. Conotoxins are small, disulfide-rich, neurotoxic peptides isolated from the marine cone snails venom. There are many types of these molecules, differing in structure and function [58]. The mechanism of their action has not been utterly elucidated yet. However, it is known that they bind to ion channels, neurotransmitters, transporters, and neural receptors [59]. Because of their selective binding to neuronal targets, conotoxins may be used for the developments of new drugs. Moreover, these peptides may be potentially used in the treatment of neurodegenerative conditions, like Alzheimer or Parkinson diseases [60]. Results of our study suggest that conotoxin may also affect the therapeutic effect of RJ, especially since some studies revealed that RJ shows a neuroprotective effect [61,62,63]. What is interesting, is that it has been investigated whether the consumption of RJ can be beneficial in the treatment of behavioral deficits in Alzheimer’s disease in rabbits [64]. Thus, the presence of conotoxin-like protein 1 in RJ may contribute to elucidate the neuroprotective effect caused by RJ. Interestingly, Kaplan et al. [65] found this protein to be mainly expressed in the bee brain. It’s also worth noting that expression of a protein with an approximate mass of 8.2 kDa, referred to as omega-conotoxin-like protein 1, has been reported by Gätschenberger et al. [66] to be induced in the haemolymph of young drones as a response to a septic injury. It may suggest that this protein participates in an immune response.
The next protein, for which the sequence similarity was noticed, was ataxin-2. This protein has been found in the BLAST analysis of gi|380026601 (Predicted: uncharacterised protein LOC100863702 [Apis florea]). However, database searching returned the BLAST scores under 100% (77% query coverage and 88.41% per cent identity). Therefore, the presence of this protein (or its homolog) in royal jelly should be confirmed in further research. Ataxin-2 is an RNA-binding protein, which regulates mRNA stability and translation. It participates in cell death, calcium homeostasis, and cellular metabolism [67,68]. In insects, ataxin-2 is involved in the regulation of circadian rhythms but also in the development of peripheral tissue [69,70,71]. This may explain the presence of ataxin-2 in RJ.
Other peptides that have been found to be similar to the sequences detected in RJ are odorant receptor Or1 and chymotrypsin inhibitor. These proteins play mainly regulatory functions. Odorant receptor Or1 targets in bee’s periphery nervous system. It participates in the odorants transport and binding, and therefore, in the behavioral response to specific odorants [72]. Chymotrypsin inhibitor prevents proteolysis of proteins present in RJ. For the chymotrypsin inhibitor, 100% similarity to gi|328783471 (Predicted: hypothetical protein LOC725114 isoform 1 [Apis mellifera]) was found. However, query coverage was lower (84%) for the analysis of gi|328783362 (Predicted: hypothetical protein LOC725249 [Apis mellifera]). Regarding odorant receptor Or1, the similarity with Predicted: uncharacterised protein LOC100864410 [Apis florea] detected in our study was expressed with 98% query coverage and 89.68% per cent identity. Therefore, these proteomic features require further research.
Other peptides for which a partial similarity to the RJ-detected proteins was found were Chorion peroxidase, regucalcin, kielin/chordin-like protein, and low-density lipoprotein receptor 1-like isoform X3. All those proteins bear mainly regulatory functions. However, due to lower BLAST scores (see Table 2), a certain identification must be proved by additional analyses. For the remaining hypothetical and uncharacterized proteins found in royal jelly, no defined references were found in BLAST analysis. The structure, functions and properties of these proteins should be investigated in future research. However, gi|380012917 (Predicted: uncharacterized protein LOC100870850 [Apis florea]), and gi|48094573 (uncharacterized protein LOC408608 [Apis mellifera]) were found in Hymenoptera venom in our previous study [56].
As the number of fully sequenced honeybee proteins and peptides increases, omics and bioinformatics technologies provide a tremendous amount of information about sequential biomolecules that function in various biological aspects of the honeybee and that are found in its products. In the postgenomic era, the international project i5K (http://i5k.github.io/.2018, accessed on 16 February 2021) not only played an important role in understanding the honeybee genome but also resulted in extensive research on the bee’s proteome and the identification of many active proteins and peptides in its products. For a number of new substances produced by honeybee, only one type of biological activity has been identified, and their wider physiological role is unknown. Because many substances often exhibit pleiotropic biological activity, there is a need to understand a wider spectrum of biological activity for newly discovered compounds. It is also necessary to develop new, specific, and sensitive physiological, pharmacological, or toxicological bioassays. In the last decade, we have been observing new trends in the development of modern biotechnology, where besides medical, industrial, food and plant protection biotechnology, the term “Yellow Biotechnology” was introduced as an alternative term for insect biotechnology, opening new horizons for multidisciplinary research in the field of experimental entomology [73]. Yellow biotechnology is defined as the use of biotechnology to transform insects, their molecules, cells or organs into products and services with great potential for applications in biomedicine, pharmacy, and industry, where the honeybee may be one of the candidates.

4. Materials and Methods

4.1. Royal Jelly

In this study, we analyzed three samples of fresh RJ, collected in June 2019. The non-commercial apiary, from which all fresh samples were collected, is located in Góry Złotnickie village (51°87′504″ N, 18°12′431″ E) in Poland (Greater Poland Voivodeship, west-central Poland). Right after the collection, samples were stored at −80 °C until analyses. All RJ samples were analyzed in three technical replicates.

4.2. Pre-Treatment of the Royal Jelly Samples

Samples of fresh royal jelly were first suspended in ultrapure water in a concentration of 330 mg/mL. The RJ-water suspensions were mixed with vortex for 1 min and then sonicated for 20 min. After sonication, suspensions were vortex-mixed for 1 min and subsequently spun with 13,000 RPM (9600 RCF) for 20 min. Collected supernatants (in this study called “extracts”) were used in further steps.

4.3. Relative Protein Enrichment—ProteoMinerTM Hexapeptide Ligand Library

For the presented research, we applied a commercial ProteoMinerTM Sequential Elution Small Capacity Kit (Bio-Rad, Hercules, CA, USA). All enrichment steps were performed strictly according to the manufacturer’s instructions. The only difference from the manufacturer’s protocol was the type of the sample solution loaded onto the ProteoMinerTM column. Instead of the 0.2 mL of serum (for which the protocol is optimized), we added 0.2 mL of RJ extract, prepared as described above (see Section 4.2). In brief, 0.2 mL of the RJ water extracts were loaded onto pre-conditioned ProteoMinerTM columns containing slurry beads and incubated at room temperature (RT) on a platform shaker. Next the samples were washed with phosphate buffer saline (PBS), and sequentially eluted with solvents: 1 M sodium chloride, 20 mM HEPES, pH 7.4 (Elution Reagent 1), 200 mM glycine, pH 2.4 (Elution Reagent 2), 60% ethylene glycol in water (Elution Reagent 3), and 33.3% 2-propanol, 16.7% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) (Elution Reagent 4). In total, 4 separate fractions were obtained from each sample. These fractions were further trypsin digested and then purified with ZipTip.

4.4. Trypsin Proteolytic Digestion and ZipTip Concentration and Purification

All RJ fractions derived from the ProteoMinerTM enrichment method and additionally crude RJ solutions (water suspended and sonicated, but not fractionated with ProteoMinerTM; three technical repetitions) were subjected to digestion with trypsin (Promega, Madison, WI, USA). We applied a modified protocol from pierce in-solution tryptic digestion kit. In brief, before adding trypsin, proteins in extracts (both ProteoMinerTM fractions and “crude” extracts non-enriched with ProteoMinerTM) were denatured, reduced, and alkylated. Subsequently, after trypsin addition, RJ extracts were digested at 37 °C overnight. In the next step, ZipTip C18 (Millipore, Bedford, MA, USA) reverse phase chromatography micropipette tips were used to purify, desalt and concentrate digested RJ protein extracts before mass spectrometry analyses. Tips were conditioned using ACN and 0.1% TFA/water. Then, each solution of the digested proteins extracted from the RJ samples (the exact solutions formed due to trypsin digestion of the RJ extracts, without any additional reagents) was loaded onto ZipTip tips according to the manufacturer’s instruction. Sample solutions were aspirated and dispensed in 10 cycles for maximum binding of proteins. After washing with 0.1% TFA, bound proteins and peptides were eluted with 50% ACN in 0.1% TFA.

4.5. NanoLC-MALDI-TOF/TOF MS/MS Analyses

For the identification of peptides and proteins included in RJ, the nanoLC-MALDI-TOF/TOF MS/MS technique was applied. Subsequent to ZipTip depletion, RJ protein extracts were subjected to nanoLC fractionation. The nanoLC set consisted of EASY-nLC II (Bruker Daltonics, Bremen, Germany) nanoflow HPLC system, and Proteineer-fc II (Bruker Daltonics, Bremen, Germany) collector of fractions. The nanoLC system parts were: NS-MP-10 BioSphere C18 (NanoSeparations, Nieuwkoop, the Netherlands) trap column (20 mm × 100 µm I.D., particle size 5 µm, pore size 120 Å), and an Acclaim PepMap 100 (Thermo Scientific, Sunnyvale, CA, USA) column (150 mm × 75 µm I.D., particle size 3 µm, pore size 100 Å). The gradient elution method was set on 2–50% of ACN in 96 min (mobile phase A—0.05% TFA in water, mobile phase B—0.05% TFA in 90% ACN). The flow rate for separation was 300 nL/min, and the volume of the sample eluent injected into the chromatography column was 4 µL. The nanoLC system was operated by HyStar 3.2 (Bruker Daltonics, Bremen, Germany) software. In total, 384 fractions were obtained in the nanoLC separation process. Fractions were mixed with a matrix solution consisted of 36 µL of HCCA saturated solution in 0.1% TFA and acetonitrile (90:10 v/v), 748 µL of acetonitrile and 0.1% TFA (95:5 v/v) mixture, 8 µL of 10% TFA, and 8 µL of 100 mM ammonium phosphate, and spotted automatically onto the AnchorChip 384 (Bruker Daltonics, Bremen, Germany) target plate by the collector of fractions. Fractions were subsequently subjected to MS/MS analysis. For this purpose, UltrafleXtreme (Bruker Daltonics, Bremen, Germany) mass spectrometer was used (mass range m/z of 700–3500, reflectron mode). External calibration was performed with a mixture of Peptide Calibration Standard (Bruker Daltonics, Bremen, Germany). The list of the precursor ions for the identification was established with WARP-LC (Bruker Daltonics, Bremen, Germany) software. For the spectra acquisition, processing and evaluation FlexControl 3.4, FlexAnalysis 3.4 and BioTools 3.2 (Bruker Daltonics, Bremen, Germany) software were used. For the identification of discriminative proteins and peptides, an NCBI database and Mascot 2.4.1 search engine with taxonomic restriction to Apis spp. were applied. The protein search parameters were as follows: fragment ion mass tolerance m/z ± 0.7, precursor ion mass tolerance ±50 ppm, peptide charge +1, monoisotopic mass.

5. Conclusions

The functional and health-promoting properties of RJ make it one of the most attractive healthy ingredients. However, because of an overly complicated composition, RJ is not yet fully characterized and may cause adverse effects. Therefore, in this study, we proposed an advanced strategy based on combinatorial hexapeptide ligand libraries (ProteoMinerTM kit) for the proteomic analysis of RJ water extracts. We identified a total of 86 proteins taxonomically classified to Apis spp. (bees). In addition to the well-known components of RJ, we identified 10 hypothetical proteins and 4 uncharacterized proteins. We also proved the utility of the ProteoMinerTM technique in the analysis of complex biological samples, as 74 out of all identified proteins were detected in RJ extracts pre-treated with ProteoMinerTM kit, and only 50 proteins were found in extracts that were not enriched with this technique. Broadening the knowledge of RJ composition may contribute to the development of standards and regulations, enhancing the quality of RJ, and consequently, the safety of its supplementation. Moreover, better characterization of proteins and peptides found in RJ may support the understanding of the functional properties of RJ regarding both humans and bees.

Author Contributions

Conceptualization, J.M. (Jan Matysiak) and G.R.; methodology, E.M. and J.M. (Jan Matysiak); software, E.M.; validation, E.M.; formal analysis, E.M. and J.M. (Joanna Matysiak); investigation, E.M.; resources, J.M. (Jan Matysiak); data curation, E.M.; Writing—Original draft preparation, E.M., J.M. (Joanna Matysiak), G.R., E.K., W.Z., J.Z. and J.M. (Jan Matysiak); Writing—Review and Editing, E.M., J.M. (Joanna Matysiak), G.R. and J.M. (Jan Matysiak); visualization, E.M.; supervision, J.M. (Jan Matysiak); project administration, J.M. (Jan Matysiak); funding acquisition, J.M. (Jan Matysiak). All authors have read and agreed to the published version of the manuscript.

Funding

This project received financial support from the Polish National Science Centre (grant number 2016/23/D/NZ7/03949).

Data Availability Statement

The data presented in this study are contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Denisow, B.; Denisow-Pietrzyk, M. Biological and therapeutic properties of bee pollen: A review. J. Sci. Food Agric. 2016, 96, 4303–4309. [Google Scholar] [CrossRef]
  2. Fratini, F.; Cilia, G.; Mancini, S.; Felicioli, A. Royal Jelly: An ancient remedy with remarkable antibacterial properties. Microbiol. Res. 2016, 192, 130–141. [Google Scholar] [CrossRef]
  3. Taulavuori, K.; Julkunen-Tiitto, R.; Hyöky, V.; Taulavuori, E. Blue Mood for Superfood. Nat. Prod. Commun. 2013, 8. [Google Scholar] [CrossRef] [Green Version]
  4. Ramadan, M.F.; Al-Ghamdi, A. Bioactive compounds and health-promoting properties of royal jelly: A review. J. Funct. Foods 2012, 4, 39–52. [Google Scholar] [CrossRef]
  5. Wytrychowski, M.; Chenavas, S.; Daniele, G.; Casabianca, H.; Batteau, M.; Guibert, S.; Brion, B. Physicochemical characterisation of French royal jelly: Comparison with commercial royal jellies and royal jellies produced through artificial bee-feeding. J. Food Compos. Anal. 2013, 29, 126–133. [Google Scholar] [CrossRef]
  6. Sabatini, A.G. Quality and standardisation of Royal Jelly. J. ApiProd. ApiMed. Sci. 2009, 1, 16–21. [Google Scholar] [CrossRef]
  7. Garcia-Amoedo, L.H.; De Almeida-Muradian, L.B. Physicochemical composition of pure and adulterated royal jelly. Quim. Nova 2007, 30, 257–259. [Google Scholar] [CrossRef]
  8. Alvarez-Suarez, J.M. Bee Products-Chemical and Biological Properties; Springer International Publishing: New York, NY, USA, 2017; pp. 1–306. [Google Scholar]
  9. Priomorac, L.; Bilić Rajs, B.; Puškadija, Z.; Kovačić, M.; Vukadin, I.; Flanjak, I. Physicochemical characteristics of Croatian royal jelly. Croat. J. Food Sci. Technol. 2019, 11, 266–271. [Google Scholar]
  10. Balkanska, R.; Crenguta, P. Comparison of physicochemical parameters in royal jelly from Romania and Bulgaria. Bull. Univ. Agric. Sci. Vet. Med. Cluj-Napoca Anim. Sci. Biotechnol. 2013, 70, 117–121. [Google Scholar]
  11. Scarselli, R.; Donadio, E.; Giuffrida, M.G.; Fortunato, D.; Conti, A.; Balestreri, E.; Felicioli, R.; Pinzauti, M.; Sabatini, A.G.; Felicioli, A. Towards royal jelly proteome. Proteomics 2005, 5, 769–776. [Google Scholar] [CrossRef]
  12. Lercker, G.; Caboni, M.F.; Vecchi, M.A.; Sabatini, A.G.; Nanetti, A. Caratterizzazione dei principali costituenti della gelatina reale. Apicoltura 1992, 8, 27–37. [Google Scholar]
  13. Sano, O.; Kunikata, T.; Kohno, K.; Iwaki, K.; Ikeda, M.; Kurimoto, M. Characterization of Royal Jelly Proteins in both Africanized and European Honeybees (Apis mellifera) by Two-Dimensional Gel Electrophoresis. J. Agric. Food Chem. 2004, 52, 15–20. [Google Scholar] [CrossRef]
  14. Buttstedt, A.; Moritz, R.F.A.; Erler, S. Origin and function of the major royal jelly proteins of the honeybee (Apis mellifera) as members of the yellow gene family. Biol. Rev. 2014, 89, 255–269. [Google Scholar] [CrossRef] [PubMed]
  15. Kamakura, M. Royalactin induces queen differentiation in honeybees. Nature 2011, 473, 478–483. [Google Scholar] [CrossRef]
  16. Buttstedt, A.; Ihling, C.H.; Pietzsch, M.; Moritz, R.F.A. Royalactin is not a royal making of a queen. Nature 2016, 537, E10–E12. [Google Scholar] [CrossRef]
  17. Fujita, T.; Kozuka-Hata, H.; Ao-Kondo, H.; Kunieda, T.; Oyama, M.; Kubo, T. Proteomic Analysis of the Royal Jelly and Characterization of the Functions of its Derivation Glands in the Honeybee. J. Proteome Res. 2013, 12, 404–411. [Google Scholar] [CrossRef] [PubMed]
  18. Schönleben, S.; Sickmann, A.; Mueller, M.J.; Reinders, J. Proteome analysis of Apis mellifera royal jelly. Anal. Bioanal. Chem. 2007, 389, 1087–1093. [Google Scholar] [CrossRef] [PubMed]
  19. Han, B.; Li, C.; Zhang, L.; Fang, Y.; Feng, M.; Li, J. Novel royal jelly proteins identified by gel-based and gel-free proteomics. J. Agric. Food Chem. 2011, 59, 10346–10355. [Google Scholar] [CrossRef] [PubMed]
  20. Furusawa, T.; Rakwal, R.; Nam, H.W.; Shibato, J.; Agrawal, G.K.; Kim, Y.S.; Ogawa, Y.; Yoshida, Y.; Kouzuma, Y.; Masuo, Y.; et al. Comprehensive royal jelly (RJ) proteomics using one- and two-dimensional proteomics platforms reveals novel RJ proteins and potential phospho/glycoproteins. J. Proteome Res. 2008, 7, 3194–3229. [Google Scholar] [CrossRef] [PubMed]
  21. Li, J.; Wang, T.; Zhang, Z.; Pan, Y. Proteomic analysis of royal jelly from three strains of western honeybees (Apis mellifera). J. Agric. Food Chem. 2007, 55, 8411–8422. [Google Scholar] [CrossRef]
  22. Hu, F.L.; Bíliková, K.; Casabianca, H.; Daniele, G.; Salmen Espindola, F.; Feng, M.; Guan, C.; Han, B.; Krištof Kraková, T.; Li, J.K.; et al. Standard methods for Apis mellifera royal jelly research. J. Apic. Res. 2017, 58, 1–68. [Google Scholar] [CrossRef] [Green Version]
  23. Collazo, N.; Carpena, M.; Nuñez-Estevez, B.; Otero, P.; Simal-Gandara, J.; Prieto, M.A. Health Promoting Properties of Bee Royal Jelly: Food of the Queens. Nutrients 2021, 13, 543. [Google Scholar] [CrossRef] [PubMed]
  24. Zhang, L.; Han, B.; Li, R.; Lu, X.; Nie, A.; Guo, L.; Fang, Y.; Feng, M.; Li, J. Comprehensive identification of novel proteins and N-glycosylation sites in royal jelly. BMC Genom. 2014, 15, 135. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Zhang, L.; Fang, Y.; Li, R.; Feng, M.; Han, B.; Zhou, T.; Li, J. Towards posttranslational modification proteome of royal jelly. J. Proteom. 2012, 75, 5327–5341. [Google Scholar] [CrossRef] [PubMed]
  26. Han, B.; Fang, Y.; Feng, M.; Lu, X.; Huo, X.; Meng, L.; Wu, B.; Li, J. In-depth phosphoproteomic analysis of royal jelly derived from western and eastern honeybee species. J. Proteome Res. 2014, 13, 5928–5943. [Google Scholar] [CrossRef] [PubMed]
  27. Greco, V.; Piras, C.; Pieroni, L.; Ronci, M.; Putignani, L.; Roncada, P.; Urbani, A. Applications of MALDI-TOF mass spectrometry in clinical proteomics. Expert Rev. Proteom. 2018, 15, 683–696. [Google Scholar] [CrossRef]
  28. Hajduk, J.; Matysiak, J.; Kokot, Z.J. Challenges in biomarker discovery with MALDI-TOF MS. Clin. Chim. Acta 2016, 458, 84–98. [Google Scholar] [CrossRef]
  29. Li, S.; He, Y.; Lin, Z.; Xu, S.; Zhou, R.; Liang, F.; Wang, J.; Yang, H.; Liu, S.; Ren, Y. Digging More Missing Proteins Using an Enrichment Approach with ProteoMiner. J. Proteome Res. 2017, 16, 4330–4339. [Google Scholar] [CrossRef]
  30. Righetti, P.G.; Boschetti, E.; Lomas, L.; Citterio, A. Protein EqualizerTM Technology: The quest for a “democratic proteome”. Proteomics 2006, 6, 3980–3992. [Google Scholar] [CrossRef]
  31. Boschetti, E.; Righetti, P.G. The ProteoMinerin the proteomic arena: A non-depleting tool for discovering low-abundance species. J. Proteom. 2008, 71, 255–264. [Google Scholar] [CrossRef]
  32. Drapeau, M.D.; Albert, S.; Kucharski, R.; Prusko, C.; Maleszka, R. Evolution of the Yellow/Major Royal Jelly Protein family and the emergence of social behavior in honey bees. Genome. Res. 2006, 16, 1385–1394. [Google Scholar] [CrossRef] [Green Version]
  33. Santos, K.S.; Delazari dos Santos, L.; Mendes, M.; Monson de Souza, B.; Malaspina, O.; Palma, M.S. Profiling the proteome complement of the secretion from hypopharyngeal gland of Africanized nurse-honeybees (Apis mellifera L.). Insect Biochem. Mol. Biol. 2005, 35, 85–91. [Google Scholar] [CrossRef]
  34. Schmitzová, J.; Klaudiny, J.; Albert, Š.; Schröder, W.; Schreckengost, W.; Hanes, J.; Júdová, J.; Šimúth, J. A family of major royal jelly proteins of the honeybee Apis mellifera L. Cell. Mol. Life Sci. 1998, 54, 1020–1030. [Google Scholar] [CrossRef]
  35. Hanes, J.; Šimuth, J. Identification and partial characterisation of the major royal jelly protein of the honey bee (Apis mellifera L.). J. Apic. Res. 1992, 31, 22–26. [Google Scholar] [CrossRef]
  36. Ramanathan, A.N.K.G.; Nair, A.J.; Sugunan, V.S. A review on Royal Jelly proteins and peptides. J. Funct. Foods 2018, 44, 255–264. [Google Scholar] [CrossRef]
  37. Buttstedt, A.; Mureşan, C.I.; Lilie, H.; Hause, G.; Ihling, C.H.; Schulze, S.H.; Pietzsch, M.; Moritz, R.F.A. How Honeybees Defy Gravity with Royal Jelly to Raise Queens. Curr. Biol. 2018, 28, 1095–1100.e3. [Google Scholar] [CrossRef] [Green Version]
  38. Pirk, C.W.W. Honeybee Evolution: Royal Jelly Proteins Help Queen Larvae to Stay on Top. Curr. Biol. 2018, 28, R350–R351. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  39. Mureşan, C.I.; Buttstedt, A. pH-dependent stability of honey bee (Apis mellifera) major royal jelly proteins. Sci. Rep. 2019, 9, 3–8. [Google Scholar] [CrossRef]
  40. Ragland, S.A.; Criss, A.K. From bacterial killing to immune modulation: Recent insights into the functions of lysozyme. PLoS Pathog. 2017, 13, e1006512. [Google Scholar] [CrossRef]
  41. Oliver, W.T.; Wells, J.E. Lysozyme as an alternative to growth promoting antibiotics in swine production. J. Anim. Sci. Biotechnol. 2015, 6, 35. [Google Scholar] [CrossRef] [Green Version]
  42. McCleskey, C.S.; Melampy, R.M. Bactericidal Properties of Royal Jelly of the Honeybee. J. Econ. Entomol. 1939, 32, 581–587. [Google Scholar] [CrossRef]
  43. Bílikova, K.; Huang, S.-C.; Lin, I.-P.; Šimuth, J.; Peng, C.-C. Structure and antimicrobial activity relationship of royalisin, an antimicrobial peptide from royal jelly of Apis mellifera. Peptides 2015, 68, 190–196. [Google Scholar] [CrossRef] [PubMed]
  44. Shen, L.; Liu, D.; Li, M.; Jin, F.; Din, M.; Parnell, L.D.; Lai, C.-Q. Mechanism of Action of Recombinant Acc-Royalisin from Royal Jelly of Asian Honeybee against Gram-Positive Bacteria. PLoS ONE 2012, 7, e47194. [Google Scholar] [CrossRef] [PubMed]
  45. Yang, Y.C.; Chou, W.M.; Widowati, D.A.; Lin, I.P.; Peng, C.C. 10-Hydroxy-2-Decenoic Acid of Royal Jelly Exhibits Bactericide and Anti-Inflammatory Activity in Human Colon Cancer Cells. BMC Complement. Altern. Med. 2018, 18, 202. [Google Scholar] [CrossRef] [PubMed]
  46. Kurek-Górecka, A.; Górecki, M.; Rzepecka-Stojko, A.; Balwierz, R.; Stojko, J. Bee Products in Dermatology and Skin Care. Molecules 2020, 25, 556. [Google Scholar] [CrossRef] [Green Version]
  47. Pavel, C.I.; Mărghitaş, L.A.; Bobiş, O.; Dezmirean, D.S.; Şapcaliu, A.; Radoi, I.; Mădaş, M.N. Biological Activities of Royal Jelly—Review. Sci. Pap. Anim. Sci. Biotechnol. 2011, 44, 108–118. [Google Scholar]
  48. Spillner, E.; Blank, S.; Jakob, T. Hymenoptera Allergens: From Venom to “Venome”. Front. Immunol. 2014, 5, 77. [Google Scholar] [CrossRef] [Green Version]
  49. Tomsitz, D.; Brockow, K. Component Resolved Diagnosis in Hymenoptera Anaphylaxis. Curr. Allergy Asthma Rep. 2017, 17, 38. [Google Scholar] [CrossRef] [PubMed]
  50. Hoffman, D.R. Hymenoptera venom allergens. Clin. Rev. Allergy Immunol. 2006, 30, 109–128. [Google Scholar] [CrossRef]
  51. Takahama, H.; Shimazu, T. Food-induced anaphylaxis caused by ingestion of royal jelly. J. Dermatol. 2006, 33, 424–426. [Google Scholar] [CrossRef]
  52. Katayama, M.; Aoki, M.; Kawana, S. Case of anaphylaxis caused by ingestion of royal jelly. J. Dermatol. 2008, 35, 222–224. [Google Scholar] [CrossRef]
  53. Mizutani, Y.; Shibuya, Y.; Takahashi, T.; Tsunoda, T.; Moriyama, T.; Seishima, M. Major royal jelly protein 3 as a possible allergen in royal jelly-induced anaphylaxis. J. Dermatol. 2011, 38, 1079–1081. [Google Scholar] [CrossRef] [PubMed]
  54. Leung, R.; Lam, C.W.K.; Ho, A.; Chan, J.K.W.; Choy, D.; Lai, C.K.W. Allergic sensitisation to common environmental allergens in adult asthmatics in Hong Kong. Hong Kong Med. J. 1997, 3, 211–217. [Google Scholar]
  55. Hata, T.; Furusawa-Horie, T.; Arai, Y.; Takahashi, T.; Seishima, M.; Ichihara, K. Studies of royal jelly and associated cross-reactive allergens in atopic dermatitis patients. PLoS ONE 2020, 15, e0233707. [Google Scholar] [CrossRef] [PubMed]
  56. Matysiak, J.; Hajduk, J.; Światły, A.; Naskret, N.; Kokot, Z.J. Proteomic analysis of Apis mellifera venom determined by liquid chromatography (LC) coupled with nano-LC-MALDI-TOF/TOF MS. Acta. Pol. Pharm. Drug Res. 2017, 74, 53–65. [Google Scholar]
  57. Matysiak, J.; Hajduk, J.; Mayer, F.; Hebeler, R.; Kokot, Z.J. Hyphenated LC–MALDI–ToF/ToF and LC–ESI–QToF approach in proteomic characterisation of honeybee venom. J. Pharm. Biomed. Anal. 2016, 121, 69–76. [Google Scholar] [CrossRef] [PubMed]
  58. Robinson, S.D.; Norton, R.S. Conotoxin gene superfamilies. Mar. Drugs 2014, 12, 6058–6101. [Google Scholar] [CrossRef] [Green Version]
  59. Daly, N.; Craik, D. Structural Studies of Conotoxins. IUBMB Life 2009, 61, 144–150. [Google Scholar] [CrossRef]
  60. Dao, F.-Y.; Yang, H.; Su, Z.-D.; Yang, W.; Wu, Y.; Hui, D.; Chen, W.; Tang, H.; Lin, H. Recent Advances in Conotoxin Classification by Using Machine Learning Methods. Molecules 2017, 22, 1057. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  61. Ahmad, S.; Campos, M.G.; Fratini, F.; Altaye, S.Z.; Li, J. New insights into the biological and pharmaceutical properties of royal jelly. Int. J. Mol. Sci. 2020, 21, 382. [Google Scholar] [CrossRef] [Green Version]
  62. Minami, A.; Matsushita, H.; Ieno, D.; Matsuda, Y.; Horii, Y.; Ishii, A.; Takahashi, T.; Kanazawa, H.; Wakatsuki, A.; Suzuki, T. Improvement of neurological disorders in postmenopausal model rats by administration of royal jelly. Climacteric 2016, 19, 568–573. [Google Scholar] [CrossRef]
  63. Mohamed, A.A.-R.; Galal, A.A.A.; Elewa, Y.H.A. Comparative protective effects of royal jelly and cod liver oil against neurotoxic impact of tartrazine on male rat pups brain. Acta Histochem. 2015, 117, 649–658. [Google Scholar] [CrossRef]
  64. Pan, Y.; Xu, J.; Jin, P.; Yang, Q.; Zhu, K.; You, M.; Hu, F.; Chen, M. Royal Jelly Ameliorates Behavioral Deficits, Cholinergic System Deficiency, and Autonomic Nervous Dysfunction in Ovariectomized Cholesterol-Fed Rabbits. Molecules 2019, 24, 1149. [Google Scholar] [CrossRef] [Green Version]
  65. Kaplan, N.; Morpurgo, N.; Linial, M. Novel Families of Toxin-like Peptides in Insects and Mammals: A Computational Approach. J. Mol. Biol. 2007, 369, 553–566. [Google Scholar] [CrossRef] [PubMed]
  66. Gätschenberger, H.; Gimple, O.; Tautz, J.; Beier, H. Honey bee drones maintain humoral immune competence throughout all life stages in the absence of vitellogenin production. J. Exp. Biol. 2012, 215, 1313–1322. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  67. Ostrowski, L.A.; Hall, A.C.; Mekhail, K. Ataxin-2: From RNA Control to Human Health and Disease. Genes 2017, 8, 157. [Google Scholar] [CrossRef] [PubMed]
  68. Carmo-Silva, S.; Nobrega, C.; Pereira de Almeida, L.; Cavadas, C. Unraveling the Role of Ataxin-2 in Metabolism. Trends Endocrinol. Metab. 2017, 28, 309–318. [Google Scholar] [CrossRef]
  69. Zhang, Y.; Ling, J.; Yuan, C.; Dubruille, R.; Emery, P. A role for Drosophila ATX2 in activation of PER translation and circadian behavior. Science 2013, 340, 879–882. [Google Scholar] [CrossRef] [Green Version]
  70. Lim, C.; Allada, R. ATAXIN-2 Activates PERIOD Translation to Sustain Circadian Rhythms in Drosophila. Science 2013, 340, 875–879. [Google Scholar] [CrossRef] [Green Version]
  71. Vianna, M.C.B.; Poleto, D.C.; Gomes, P.F.; Valente, V.; Paçó-Larson, M.L. Drosophila ataxin-2 gene encodes two differentially expressed isoforms and its function in larval fat body is crucial for development of peripheral tissues. FEBS OpenBio 2016, 6, 1040–1453. [Google Scholar] [CrossRef]
  72. Venthur, H.; Zhou, J.-J. Odorant Receptors and Odorant-Binding Proteins as Insect Pest Control Targets: A Comparative Analysis. Front. Physiol. 2018, 9, 1163. [Google Scholar] [CrossRef] [PubMed]
  73. Vilcinskas, A. Biotechnology I Insect Biotechnologie in Drug Discovery, 1st ed.; Vilcinskas, A., Ed.; Springer: Berlin/Heidelberg, Germany, 2013; p. 201. [Google Scholar]
Molecules 26 02762 g001 550
Figure 1. The number of proteins identified in unpurified samples, samples pre-treated with ProteoMinerTM, and both in unpurified and purified samples.
Figure 1. The number of proteins identified in unpurified samples, samples pre-treated with ProteoMinerTM, and both in unpurified and purified samples.
Molecules 26 02762 g001
Molecules 26 02762 g002 550
Figure 2. Classes of proteins identified (A) in total in all RJ “crude” and pre-treated; (B) only in crude (non-ProteoMinerTM pre-treated) RJ samples; (C) only in ProteoMinerTM pre-treated RJ samples.
Figure 2. Classes of proteins identified (A) in total in all RJ “crude” and pre-treated; (B) only in crude (non-ProteoMinerTM pre-treated) RJ samples; (C) only in ProteoMinerTM pre-treated RJ samples.
Molecules 26 02762 g002
Table
Table 1. List of proteins identified in royal jelly found in: CRJ—“crude” (non-enriched with ProteoMinerTM technique) water extracts of royal jelly; F1, F2, F3, F4—separate fractions obtained from ProteoMinerTM elution with 1 M sodium chloride, 20 mM HEPES, pH 7.4 (F1), 200 mM glycine, pH 2.4 (F2), 60% ethylene glycol in water (F3), and 33.3% 2-propanol, 16.7% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) (F4); MW—molecular weight; pI—isoelectric point. Fractions, in which proteins were detected, marked with “x”.
Table 1. List of proteins identified in royal jelly found in: CRJ—“crude” (non-enriched with ProteoMinerTM technique) water extracts of royal jelly; F1, F2, F3, F4—separate fractions obtained from ProteoMinerTM elution with 1 M sodium chloride, 20 mM HEPES, pH 7.4 (F1), 200 mM glycine, pH 2.4 (F2), 60% ethylene glycol in water (F3), and 33.3% 2-propanol, 16.7% acetonitrile (ACN), 0.1% trifluoroacetic acid (TFA) (F4); MW—molecular weight; pI—isoelectric point. Fractions, in which proteins were detected, marked with “x”.
AccessionProtein NameFunctionMW [kDa]pICRJF1F2F3F4
gi|110749126Predicted: glucose dehydrogenase [acceptor] isoform 3 [Apis mellifera]Enzyme70.16.7 x
gi|110751029Predicted: e3 ubiquitin-protein ligase IAP-3-like [Apis mellifera]Enzyme43.26.4 x
gi|110756431Predicted: hypothetical protein LOC725074 [Apis mellifera] 8.310.2x
gi|110758964Predicted: regucalcin-like [Apis mellifera]Binding10.29.4x x
gi|110763647Predicted: hypothetical protein LOC726323 [Apis mellifera] 18.57.9 x
gi|13184963defensin [Apis mellifera]Defense6.36.5 x
gi|166795901apolipophorin-III-like protein precursor [Apis mellifera]Binding21.35.4xxxxx
gi|202078658defensin [Apis cerana cerana]Defense10.67.8x
gi|202078660defensin [Apis cerana cerana]Defense10.77.8 x
gi|254548151defensin precursor [Apis cerana]Defense8.76.4xxxxx
gi|254548155defensin precursor [Apis mellifera]Defense8.85.9xxxxx
gi|254910938defensin-1 preproprotein [Apis mellifera]Defense10.76.4 x
gi|258678306MRJP9 [Apis cerana]Honeybee nutrition and development19.49.2 x
gi|258678310MRJP5 [Apis dorsata]Honeybee nutrition and development21.34.7x
gi|258678314MRJP6 [Apis florea]Honeybee nutrition and development21.54.4 xx
gi|258678316MRJP9 [Apis florea]Honeybee nutrition and development21.05.5xxxxx
gi|283105164alpha-glucosidase III [Apis dorsata]Enzyme65.55.0x x
gi|284182838major royal jelly protein 4 [Apis mellifera]Honeybee nutrition and development53.05.9xxxx
gi|284812514MRJP5 [Apis mellifera]Honeybee nutrition and development70.16.1xxxxx
gi|288872651major royal jelly protein [Apis mellifera]Honeybee nutrition and development61.66.7xxxxx
gi|28972896major royal jelly protein-like protein [Apis dorsata]Honeybee nutrition and development9.29.8xxxxx
gi|328775853Predicted: DNA replication licensing factor MCM4-like [Apis mellifera]Binding80.76.9 x
gi|328777366Predicted: hypothetical protein LOC100577348 [Apis mellifera] 61.110.5 x
gi|328779534Predicted: hypothetical protein LOC552041 [Apis mellifera] 79.34.2 x
gi|328782858Predicted: hypothetical protein LOC410515 [Apis mellifera] 190.26.4x
gi|328783362Predicted: hypothetical protein LOC725249 [Apis mellifera] 6.34.9x
gi|328783471Predicted: hypothetical protein LOC725114 isoform 1 [Apis mellifera] 10.15.7x
gi|328784821Predicted: hypothetical protein LOC100577210 [Apis mellifera] 11.65.9xx xx
gi|328789019Predicted: protein SERAC1-like [Apis mellifera]Remodeling87.59.2 x
gi|328790726Predicted: venom acid phosphatase Acph-1-like [Apis mellifera]Enzyme42.68.5 x
gi|328792767Predicted: hypothetical protein LOC724993 [Apis mellifera] 43.85.4 x
gi|328793775Predicted: s-adenosylmethionine decarboxylase proenzyme-like, partial [Apis mellifera]Enzyme32.14.6x
gi|328794347Predicted: major royal jelly protein 3-like, partial [Apis mellifera]Honeybee nutrition and development42.76.5x x
gi|33358394major royal jelly protein MRJP1 [Apis cerana cerana]Honeybee nutrition and development49.05.4xxxxx
gi|380011960Predicted: slit homolog 2 protein-like [Apis florea]Binding155.36.0 x
gi|380012917Predicted: uncharacterized protein LOC100870850 [Apis florea] 18.97.2 x
gi|380013532Predicted: Low Quality Protein: clathrin heavy chain-like [Apis florea]Binding187.75.6 x
gi|380016522Predicted: probable bifunctional methylenetetrahydrofolate
dehydrogenase/cyclohydrolase 2-like [Apis florea]		Enzyme39.99.6 x
gi|380017034Predicted: glucosylceramidase-like [Apis florea]Enzyme60.85.5 xx
gi|380019073Predicted: lysozyme 1-like isoform 2 [Apis florea]Enzyme13.84.6x xx
gi|380020436Predicted: regucalcin-like [Apis florea]Binding37.75.3xx xx
gi|380022658Predicted: major royal jelly protein 3-like [Apis florea]Honeybee nutrition and development62.77.8xx
gi|380022660Predicted: major royal jelly protein 4-like [Apis florea]Honeybee nutrition and development56.16.1 x
gi|380022665Predicted: major royal jelly protein 1-like [Apis florea]Honeybee nutrition and development43.95.3xxxxx
gi|380022667Predicted: major royal jelly protein 2-like [Apis florea]Honeybee nutrition and development49.15.7xx
gi|380022669Predicted: major royal jelly protein 2-like [Apis florea]Honeybee nutrition and development49.36.0xxxxx
gi|380022673Predicted: major royal jelly protein 5-like [Apis florea]Honeybee nutrition and development34.24.7 x
gi|380022681Predicted: major royal jelly protein 5-like isoform 2 [Apis florea]Honeybee nutrition and development47.59.2 x
gi|380023404Predicted: uncharacterized protein LOC100869599 [Apis florea] 18.79.5x xx
gi|380024584Predicted: chymotrypsin inhibitor-like [Apis florea]Enzyme inhibitor5.610.0x
gi|380024588Predicted: chymotrypsin inhibitor-like [Apis florea]Enzyme inhibitor8.05.0x
gi|380025248Predicted: alkylated DNA repair protein alkB homolog 8-like [Apis florea]Enzyme68.29.3xx
gi|380025500Predicted: venom acid phosphatase Acph-1-like [Apis florea]Enzyme39.94.9 x
gi|380025661Predicted: glucose dehydrogenase [acceptor]-like [Apis florea]Enzyme67.96.4xx xx
gi|380026601Predicted: uncharacterized protein LOC100863702 [Apis florea] 9.95.0 xx
gi|380027252Predicted: uncharacterized protein LOC100864410 [Apis florea] 33.49.7 x
gi|380028593Predicted: Low Quality Protein: delta-1-pyrroline-5-carboxylate
synthase-like [Apis florea]		Enzyme84.99.1 x
gi|40218299major royal jelly protein MRJP5 [Apis cerana cerana]Honeybee nutrition and development70.58.8 x
gi|40218301major royal jelly protein MRJP2 [Apis cerana cerana]Honeybee nutrition and development53.09.1x x x
gi|40557703major royal jelly protein MRJP1 precursor [Apis cerana]Honeybee nutrition and development48.95.4xxxxx
gi|40557705major royal jelly protein MRJP2 precursor [Apis cerana]Honeybee nutrition and development52.58.9xxxxx
gi|42601246major royal jelly protein MRJP5 precursor [Apis cerana]Honeybee nutrition and development68.29.3 x x
gi|46358503major royal jelly protein 2 [Apis cerana]Honeybee nutrition and development52.48.9xxxxx
gi|48094573Predicted: hypothetical protein LOC408608 [Apis mellifera] 19.47.5 xx
gi|48101366Predicted: venom serine protease 34 [Apis mellifera]Enzyme44.65.9 x
gi|562090defensin precursor [Apis mellifera]Defense10.76.4 xx
gi|56422035major royal jelly protein 3 [Apis mellifera carnica]Honeybee nutrition and development65.77.1xxxxx
gi|56422037major royal jelly protein 3 [Apis cerana]Honeybee nutrition and development69.29.3xx x
gi|56422041major royal jelly protein 3 [Apis florea]Honeybee nutrition and development59.36.4x
gi|57546160major rojal jelly protein 7 [Apis cerana]Honeybee nutrition and development24.95.5xxxxx
gi|58585090glucose oxidase [Apis mellifera]Enzyme67.96.5 xx
gi|58585098major royal jelly protein 1 precursor [Apis mellifera]Honeybee nutrition and development48.95.0xxxxx
gi|58585108major royal jelly protein 2 precursor [Apis mellifera]Honeybee nutrition and development51.07.0xxxxx
gi|58585138major royal jelly protein 5 precursor [Apis mellifera]Honeybee nutrition and development70.25.9x
gi|58585142major royal jelly protein 3 precursor [Apis mellifera]Honeybee nutrition and development61.66.5x x x
gi|58585164alpha-glucosidase precursor [Apis mellifera]Enzyme65.54.9 x
gi|58585170major royal jelly protein 4 precursor [Apis mellifera]Honeybee nutrition and development52.95.9xxxxx
gi|58585188major royal jelly protein 6 precursor [Apis mellifera]Honeybee nutrition and development49.85.9xxxxx
gi|60115688icarapin-like precursor [Apis mellifera]Venom carbohydrate-rich protein24.84.4xx xx
gi|62198227major royal jelly protein 7 precursor [Apis mellifera]Honeybee nutrition and development50.54.8xxxxx
gi|66504790Predicted: integrator complex subunit 7-like isoform 1 [Apis mellifera]snRNA processing105.99.5x
gi|66511554Predicted: glucosylceramidase-like isoform 1 [Apis mellifera]Enzyme59.35.2xxxxx
gi|66564326Predicted: plasma glutamate carboxypeptidase-like isoform 1 [Apis mellifera]Enzyme52.95.1 x
gi|66565246Predicted: lysozyme isoform 1 [Apis mellifera]Enzyme17.14.6 xx
gi|76496465major royal jelly protein 3 [Apis dorsata]Honeybee nutrition and development66.96.6xxxxx
gi|94471624icarapin variant 2 precursor [Apis mellifera]Venom carbohydrate-rich protein19.64.2 x
Table
Table 2. Results of BLAST analysis.
Table 2. Results of BLAST analysis.
AccessionProtein NameSignificant Alignment SequenceQuery CoveragePercent IdentityE-Value
gi|110756431Predicted: hypothetical protein LOC725074 [Apis mellifera]omega-conotoxin-like protein 1 [Apis mellifera]100%100.00%2.00 × 10−47
gi|110763647Predicted: hypothetical protein LOC726323 [Apis mellifera]uncharacterized protein LOC726323 isoform X1 [Apis mellifera]100%99.39%1.00 × 10−116
gi|328777366Predicted: hypothetical protein LOC100577348 [Apis mellifera]uncharacterized protein LOC100577348 isoform X2 [Apis mellifera]98%98.37%0.0
gi|328779534Predicted: hypothetical protein LOC552041 [Apis mellifera]uncharacterized protein LOC102655185 isoform X1 [Apis mellifera]100%71.55%5.00 × 10−92
gi|328782858Predicted: hypothetical protein LOC410515 [Apis mellifera]uncharacterized protein LOC410515 [Apis mellifera]93%100.00%0.0
chorion peroxidase [Habropoda laboriosa]93%62.86%0.0
gi|328783362Predicted: hypothetical protein LOC725249 [Apis mellifera]chymotrypsin inhibitor-like [Apis mellifera]84%100.00%1.00 × 10−25
gi|328783471Predicted: hypothetical protein LOC725114 isoform 1 [Apis mellifera]chymotrypsin inhibitor [Apis mellifera]100%100.00%2.00 × 10−59
gi|328784821Predicted: hypothetical protein LOC100577210 [Apis mellifera]uncharacterized protein LOC413627 [Apis mellifera]77%100.00%6.00 × 10−45
regucalcin [Apis cerana cerana]77%94.81%4.00 × 10−45
gi|328792767Predicted: hypothetical protein LOC724993 [Apis mellifera]uncharacterized protein LOC724993 [Apis mellifera]83%90.37%0.00 × 10+00
Predicted: kielin/chordin-like protein [Trachymyrmex zeteki]82%45.91%2.00 × 10−85
kielin/chordin-like protein [Nasonia vitripennis]81%47.06%3.00 × 10−89
gi|380012917Predicted: uncharacterized protein LOC100870850 [Apis florea]uncharacterized protein LOC408608 [Apis mellifera]99%73.33%2.00 × 10−84
gi|380023404Predicted: uncharacterized protein LOC100869599 [Apis florea]uncharacterized protein LOC102654257 [Apis mellifera]100%94.71%7.00 × 10−116
low-density lipoprotein receptor 1-like isoform X3 [Vespa mandarinia]98%55.88%7.00 × 10−57
gi|380026601Predicted: uncharacterized protein LOC100863702 [Apis florea]ataxin-2 [Apis cerana cerana]77%88.41%5.00 × 10−30
gi|380027252Predicted: uncharacterized protein LOC100864410 [Apis florea]odorant receptor Or1 [Apis mellifera]98%89.68%0.0
gi|48094573Predicted: hypothetical protein LOC408608 [Apis mellifera]uncharacterized protein LOC408608 [Apis mellifera]100%100.00%3.00 × 10−124
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Matuszewska, E.; Matysiak, J.; Rosiński, G.; Kędzia, E.; Ząbek, W.; Zawadziński, J.; Matysiak, J. Mining the Royal Jelly Proteins: Combinatorial Hexapeptide Ligand Library Significantly Improves the MS-Based Proteomic Identification in Complex Biological Samples. Molecules 2021, 26, 2762. https://doi.org/10.3390/molecules26092762

AMA Style

Matuszewska E, Matysiak J, Rosiński G, Kędzia E, Ząbek W, Zawadziński J, Matysiak J. Mining the Royal Jelly Proteins: Combinatorial Hexapeptide Ligand Library Significantly Improves the MS-Based Proteomic Identification in Complex Biological Samples. Molecules. 2021; 26(9):2762. https://doi.org/10.3390/molecules26092762

Chicago/Turabian Style

Matuszewska, Eliza, Joanna Matysiak, Grzegorz Rosiński, Elżbieta Kędzia, Weronika Ząbek, Jarosław Zawadziński, and Jan Matysiak. 2021. "Mining the Royal Jelly Proteins: Combinatorial Hexapeptide Ligand Library Significantly Improves the MS-Based Proteomic Identification in Complex Biological Samples" Molecules 26, no. 9: 2762. https://doi.org/10.3390/molecules26092762

APA Style

Matuszewska, E., Matysiak, J., Rosiński, G., Kędzia, E., Ząbek, W., Zawadziński, J., & Matysiak, J. (2021). Mining the Royal Jelly Proteins: Combinatorial Hexapeptide Ligand Library Significantly Improves the MS-Based Proteomic Identification in Complex Biological Samples. Molecules, 26(9), 2762. https://doi.org/10.3390/molecules26092762

Article Metrics

Back to TopTop