Quantification of Major Royal Jelly Protein 1 in Fresh Royal Jelly by

Article Cite This: J. Agric. Food Chem. 2018, 66, 1270−1278

pubs.acs.org/JAFC

Quantification of Major Royal Jelly Protein 1 in Fresh Royal Jelly by Ultraperformance Liquid Chromatography−Tandem Mass Spectrometry Na Lin,† Si Chen,§,⊥ Hong Zhang,† Junmin Li,† and Linglin Fu*,‡ †

School of Food Science and Biological Engineering and ‡Food Safety Key Laboratory of Zhejiang Province, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang 310018, China § Key Laboratory of Mariculture & Enhancement, Marine Fisheries Research Institute of Zhejiang Province, Zhoushan 316000, China ⊥ Marine and Fisheries Research Institute, Zhejiang Ocean University, Zhoushan, Zhejiang 316000, China S Supporting Information *

ABSTRACT: Major royal jelly protein 1 (MRJP1) is the most abundant protein in royal jelly (RJ), and the level of MRJP1 has been suggested as a promising parameter for standardization and evaluation of RJ authenticity in quality. Here, a quantitative method was developed for the quantification of MRJP1 in RJ based on a signature peptide and a stable isotope-labeled internal standard peptide FFDYDFGSDER*(R*, 13C6, 15N4) by ultraperformance liquid chromatography−tandem mass spectrometry. Recoveries of the established method ranged from 85.33 to 95.80%, and both the intra- and interday precision were RSD < 4.97%. Quantification results showed that content of MRJP1 in fresh RJ was 41.96−55.01 mg/g. Abnormal levels of MRJP1 were found in three commercial RJs and implied that these samples were of low quality and might be adulterated. Results of the present work suggested that the developed method could be successfully applied to quantify MRJP1 in RJ and also could evaluate the quality of RJ. KEYWORDS: MRJP1, royal jelly, quantitative method, signature peptide, stable isotope-labeled internal standard peptide, UPLC−MS/MS

1. INTRODUCTION Royal jelly (RJ) is a milky secretion derived from the hypopharyngeal and mandibular glands of young worker bees (Apis mellifera L.) and is used to feed the larvae.1 The composition of RJ is complex, and it contains water, proteins, sugars, lipids, free amino acids, and vitamins.2 RJ is one of the most attractive ingredients for healthy foods and is extensively used as cosmetic or dietary supplement due to its bioactivities and pharmacological activities.3 About 80−90% of RJ proteins are major royal jelly proteins (MRJPs),4 and nine members of MRJP1−9 have been reported.5,6 MRJPs are thought to be major factors responsible for the specific physiological role of RJ.7,8 MRJP1, designated as apalbumin 1, constitutes 45% of watersoluble proteins and is the most abundant protein in RJ.9 It is a weak acidic glycoprotein (molecular weight of its monomer is 55 kDa) and forms an oligomer that is estimated to be 350 or 420 kDa.10 It has been reported that some biological effects can be attributed to MRJP1.8,11−13 It exhibits antihypertensive activity,12 enhances proliferation,8 and also regulates mouse macrophages to release tumor necrosis factor α (TNF-α).13,14 Simuth found that MRJP1 could serve as an important factor participating in the processing of honeybee products4 because mRNA for MRJP1 was detected in hypopharyngeal glands of foragers.15 Given these data mentioned above, we propose that the quantification of MRJP1 could be used as a criterion for the evaluation of authenticity of honeybee products such as RJ and honey. Also, these data strongly suggest that the MRJP1 level might become a new quality index for RJ. © 2018 American Chemical Society

High performance liquid chromatography analysis of MRJP1 separated from RJ with column chromatography has been used primarily to determine the content of MRJP1 in RJ.16 Recently, enzyme-linked immune sorbent assay (ELISA) using purified MRJP1 with polyclonal antibody against recombinant MRJP1 was used to measure MRJP1 in honey,17 but the method was queried by Shen, who designed a MRJP1-specific peptide to determine the freshness in RJ by ELISA.18 Yamaguchi et al. were the first to determine MRJP1 levels in RJ using specific MRJP1 antibody;19 however, the novel method lacked the essential information such as how to design MRJP1-specific peptide and purify the antibody. Liquid chromatography−mass spectrometer (LC−MS) technique is increasingly contributing to the accurate characterization, identification, and quantification of proteins in complex biological matrices because of its high selectivity and sensitivity.20−22 The protein quantification by LC−MS can be achieved by detecting the entire proteins or signature peptides after trypsin digestion.23 The strategy of peptide level can meet the quantification requirements by analyzing a tryptic signature peptide whose sequence is specific for the targeted protein. The key points of the methodology are to select one or more specific signature peptides for the targeted protein and a suitable internal standard peptide. On the basis of the strategy mentioned above, we want to design a Received: Revised: Accepted: Published: 1270

December 5, 2017 January 22, 2018 January 22, 2018 January 30, 2018 DOI: 10.1021/acs.jafc.7b05698 J. Agric. Food Chem. 2018, 66, 1270−1278

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Journal of Agricultural and Food Chemistry Table 1. Parameters of MRM for Targeted Peptides protein

sequence

signature peptide

FFDYDFGSDER

isotope-labeled IS

precursor ion

cone voltage (V)

699.5

35

704.2

35

FFDYDFGSDER*

collision energy (V)

product ions

ions type

22 22 22 25 23 22

1103.3 825.2 563.2 1113.2 835.2 573.2

y9 y7 y5 y9 y7 y5

mobile phase A (0.1% FA, 2% ACN/H2O) and delivered at a flow rate of 2 μL/min. The electrospray interface was set in positive ionization mode with a spray voltage at 2.4 kV, curtain gas of 30 psi, nebulizing gas of 5 psi, and a heating temperature at 150 °C. The scan mode was IDA (information-dependent analysis). Data acquisition and processed were performed using Protein Pilot Software v. 4.5 (AB SCIEX, USA). The database was generated from protein sequences of honeybee. The retrieval parameters were set as follows: tryptic cleavage specificity was set for up to two missed cleavages, carbamidomethyl as a fixed modification, and deamidation and oxidation as the only variable modifications for the RJ samples. The false discovery rate (FDR) was controlled to 1%. All the identified peptides were manually checked by applying the cutoff criteria: protein unused score >1.3, confidence of peptide >95%. 2.5. Preparation of Tryptic Hydrolysates of MRJP1. Protein content of the supernatant obtained in section 2.2 was evaluated by Coomassie brilliant blue G-250.26 Fifty microliters of the protein supernatant was diluted with 50 mM NH4HCO3 to a final concentration of total protein at approximately 2 mg/mL. Then 500 μL of sample solution was spiked with 200 μL of 1 μg/mL internal standard peptide (FFDYDFGSDER*) and mixed with additional 50 mM NH4HCO3. The mixture was reduced by adding 10 μL of 500 mM DTT solution for 30 min at 56 °C and then alkylated with 30 μL of 500 mM IAA solution for 30 min at room temperature in the dark. Subsequently, trypsin solution (1:50, w/w, enzyme to substrate ratio) was added to the RJ sample and incubated overnight in 37 °C water bath. When the reaction finished, 5 μL of FA was added to deactivate trypsin. The digested mixture was then diluted with the initial mobile phase (H2O/ACN = 97:3, v/v, with 0.1% FA) to 2 mL and centrifuged at 12 000g for 20 min. The supernatant was passed through a 0.22 μm filter before analysis by ultraperformance liquid chromatography− tandem mass spectrometry (UPLC−MS/MS). 2.6. Synthesis of Peptide Standards. The signature peptide FFDYDFGSDER (corresponding to amino acid residues 39−49 of MRJP1) and stable isotope-labeled internal standard (IS) peptide FFDYDFGSDER* (R*, Arg-OH-13C6, 15N4) were synthesized by ChinaPeptides Co., Ltd. (Shanghai, China) with purity of more than 95%. 2.7. Preparation of Standards Stock Solutions. Stock solutions (1 mg/mL) of signature peptide FFDYDFGSDER and isotope-labeled internal standard peptide FFDYDFGSDER* were prepared by dissolving in the following order: 500 μL of ACN and then 500 μL of water. These solutions were subpacked and stored at −20 °C until for analysis. A series of signature peptide standards (5, 10, 20, 100, 250, 500, 800, and 1000 ng/mL) was prepared in the initial mobile phase (97:3 v/v, water/ACN, with 0.1% formic acid), followed by addition of 100 ng of IS peptide. Calibration curve was constructed by plotting analyte/IS peptide peak area ratio versus analyte/IS concentration ratio. 2.8. UPLC−MS/MS Analysis. The preliminary analysis and identification of the specific signature peptides in the tryptic RJ samples were performed on Q-TOF mass analyzer (Waters XEVO G2S, Waters Corporation, USA). The chromatographic separation was performed at 40 °C with a Waters ACQUITY UPLC system. The peptides separation was accomplished on an Acquity BEH C18 column (1.7 μm, 2.1 × 100 mm2, Waters Corporation, MA, USA). MS/MS was performed on triple-quadrupole mass spectrometer with an electrospray ionization

signature peptide, which is only specific for MRJP1, and select a stable internal standard (IS) peptide for the accurate and sensitive quantification of this dominant protein in RJ and other honeybee products. To the best of our knowledge, so far, neither official and confirmatory methods nor certified reference materials could be used to support a harmonized approach to the quantitative analysis of MRJP1 in royal jelly. The aim of this paper is to develop a new reliable quantitative method to quantify MRJP1 in fresh RJ samples and use this method for quality control and authenticity identification of RJ in production and commerce.

2. MATERIALS AND METHODS 2.1. Materials. Dithiotheritol (DTT), iodoacetamide (IAA), and ammonium bicarbonate (NH4HCO3) were analytical grade and obtained from Sigma-Aldrich. Formic acid (FA) of HPLC grade was provided by Tedia (purity 96%, Fairfield, USA). Acetonitrile (ACN) was of HPLC grade and purchased from Merck (Darmstadt, Germany). Sequencing grade trypsin (specific activity, > 8000 units/ mg protein) was purchased from Sigma-Aldrich. Ultrapure water was obtained from a Milli-Q purification system (Millipore, MA, USA). Electrophoresis-grade acrylamide, N,N′-methylene-bis-acrylamide, glycine, N,N,N′,N′-tetraethylmethylenediamine (TEMED), ammonium persulfate (APS), Tris-HCl, sodium dodecyl sulfate (SDS), and glycine were from Bio-Rad. Twenty-one fresh RJ samples were kindly provided from Quzhou Entry-Exit Inspection and Quarantine Bureau (Zhejiang, China). The other seven RJ samples and two honey samples were purchased from local markets in China. All samples were stored at −20 °C until analysis. 2.2. Extraction of Proteins. Appropriate RJ was diluted in deionized water by a ratio of 4:5 (w/w), stirred at room temperature for 20 min, and centrifuged at 12 000g at 4 °C for 50 min. Then the supernatant was collected and stored at −20 °C until further analysis. Contents of proteins in RJ were obtained using the Micro-Kjeldahl method24 with a conversion factor of 6.25. 2.3. SDS-PAGE Analysis of RJ. RJ protein solutions were properly diluted and then subjected to SDS-PAGE.25 MRJP1 was separated form other proteins by using a 12% separating gel and a 5% stacking gel, and then stained with Coomassie blue. 2.4. Identification of MRJP1 in RJ. The targeted protein bands were excised from the electrophoretic gel and washed with 50% ACN and 25 mM NH4HCO3. The wash was discarded and replaced by ACN to dehydrate the excised gel bands. Then 10 mM DTT was added, and the proteins were reduced at 56 °C for 1 h. After cooling to room temperature, 50 mM IAA was added, and the proteins were alkylated for 15 min at room temperature in the dark. The gels were then washed by water and dehydrated by ACN. To the dried gel pieces, trypsin (1:4, w/w enzyme to substrate ratio) in 25 mM NH4HCO3 was added and incubated at 37 °C overnight. Peptides were extracted from the gel by removing the incubation buffer and using 67% ACN containing 5% TFA in water with incubation at 37 °C for 30 min and then with sonication for 15 min. All extracts were pooled and lyophilized prior to LC−MS/MS analysis. The lyophilized peptides were sequenced and identified by NanoRPLC-Triple TOF 5600. The analytical column is C18 (75 μm × 15 cm, 3 μm, 120 μm, ChromXP Eksigent). Peptides were redissolved in 1271

DOI: 10.1021/acs.jafc.7b05698 J. Agric. Food Chem. 2018, 66, 1270−1278

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Journal of Agricultural and Food Chemistry source operated in positive-ion mode. The capillary voltage was 3.5 kV, cone voltage of 35 V, extractor voltage of 3 V, RF lens of 0.3 V, source temperature of 120 °C, desolvation temperature of 380 °C, desolvation gas flow of 600 L/h, and cone gas flow of 50 L/h. Instrument control, data acquisition, and the processing were performed using MassLynx 4.1 software. The UPLC−MS/MS condition was set as follows: the injection volume was 10 μL and the flow rate was 0.3 mL/min. The mobile phase consisted of 0.1% formic acid aqueous solution (Solvent A) and ACN with 0.1% formic acid (Solvent B). Elution started with gradient of 97% A to 60% A in 10 min, then from 60% A to 100% A in 0.1 min and kept at 100% A for 1 min, followed by a gradient to 97% A in 1 min. The column was then equilibrated at 97% A for 3 min before the next injection. Targeted peptides were detected by multiple reaction monitoring (MRM) mode. Mass transitions monitored in the method were shown in Table 1. 2.9. Method Validation. The established method was validated in terms of its sensitivity, linearity, recovery, precision, and reproducibility. Sensitivity was evaluated by the limit of detection (LOD) and limit of quantification (LOQ). LOD and LOQ of the analytical method were determined as the concentrations of the targeted MRJP1 equivalent to 3- and 10-times the signal-to-noise (S/N) on analysis of tryptic hydrolysates, respectively. The linearity of the standard curve with nine concentration points in the range of 5−1000 ng/mL spiked with a fixed concentration of IS peptide obtained by the internal standard method. The reproducibility was evaluated by calculating intra- and interday precision and accuracy, expressed as RSD (%) values and recoveries obtained from the spiked samples. Recovery experiments were performed with RJ samples spiked with three different concentrations of signature peptide and a fixed concentration of IS peptide. Recovery rate (%) was calculated as the result of the measured value minus the original level in sample divided by the spiked value. Intraday (n = 9) and interday (n = 9) precisions were evaluated by analysis of spiked samples at different times on the same day and on the consecutive days, respectively.

Figure 1. SDS-PAGE of RJ protein. From left to right: lane 1, protein marker; lane 2 and lane 3, RJ proteins. Band 1, MRJP1; band 2, MRJP2.

for developing LC−MS/MS method for protein quantification. The candidate peptides are selected based on the following principles: the sequence of peptides should not be too long or too short (8−20 amino acid is appropriate), the signature peptides should be specific to the targeted protein and can be reproducibly observed between sample preparations, they should not contain amino acids susceptible to chemical modification, the relative intensity of their MS signal and the abundance of their ions in tryptic raw royal jelly should be high enough, they should not have missed cleavage sites for trypsin, and the selected peptides should have stable physical and chemical properties. In addition, the signature peptides of RJ were obtained by comparing the theoretical and endogenous peptides. On the basis of the principles mentioned above, three tryptic peptides reproducibly detectable by UPLC−MS/MS analysis in the tryptic products of RJ samples were selected in the initial experiment, and their information was shown in Table S1. The three candidate peptides during LC−MS/MS (Q-TOF) analysis showed charged states and corresponding molecular weights in good agreement with their theoretical values. The singly charged ions of the three peptides were m/z 1377.7362, 1300.6077, and 1397.5610. Similarly, the doubly charged ions of the peptides fragments were m/z 689.3738, 650.8079, and 699.2865, and their corresponding peptides sequences were YNGVPSSLNVISK, LTSNTFDYDPK, and FFDYDFGSDER (Figure 2). Specificity of the three candidate peptides was validated by BLAST search in UniProt (www.uniprot.org). The other eight major royal jelly proteins MRJP2−9, who share a common evolutionary ancestor of the yellow protein from Drosophilia melanogaster,29 and their protein sequences were aligned with that of MRJP1 using the Clustal Omega program (www. uniprot.org/align). The alignment of amino acid sequences of the nine members of the MRJP family (Figure S1) showed high homology among them, and sequences of YNGVPSSLNVISK and FFDYDFGSDER were only specific for MRJP1. While sequence of LTSNTFDYDPK belongs to both MRJP1 (corresponding to amino acid residues 226−236) and MRJP5 (corresponding to amino acid residues 227−237), whose amino acid sequence is not unique to MRJP1 and could be eliminated from the candidate peptides. Tryptic peptides tend to be doubly charged during MS analysis (especially in an acidic solution) because trypsin cleaves peptides at the carboxyterminal side of lysine (K) and arginine (R).30 In the MS analysis, doubly charged tryptic peptides were preferred than

M1

X=

φcV M 2 m

2.10. Calculation Formula of MRJP1 Content in Samples. Where X (mg/g) is the content of MRJP1 in fresh RJ samples, m (g) is the content of RJ samples, φ is the dilution ratio of the RJ proteins, c (ng/mL) is the concentration of signature peptide in RJ samples, V (mL) is the volume of the trypsin digests, and M1/M2 is the molar mass ratio of MRJP1 and the signature peptide. 2.11. Statistics/Data Analysis. Experimental data were presented as the mean values ± SD. The statistics analysis was carried out with Microsoft Excel 2016. Data acquisition and processed of Triple TOF 5600 were performed using Protein Pilot Software v. 4.5. Mass instrument control, data acquisition, and the processing of Q-TOF and QQQ were performed using MassLynx 4.1 software.

3. RESULTS AND DISCUSSION 3.1. Identification of Major Royal Jelly Protein 1 (MRJP1) in RJ. As shown in Figure 1, molecular weights of 45−70 kDa were the major proteins in RJ, and bands 1 and 2 were the most abundant proteins. According to the content and molecular weight, we can infer that band 1 and band 2 were MRJP1 and MRJP2, respectively.27,28 Bands 1 and 2 were excised and in-gel digested by trypsin, and then the targeted proteins were characterized by LC−MS/MS. As expected, proteins of band 1 and band 2 were identified as MRJP1 (a total of 298 peptides were identified in the tryptic fragments of band 1 and peptides coverage was 88.89%) and MRJP2 (peptides coverage, 80.53%), respectively, by nano-RPLCTriple TOF 5600. 3.2. Selection and Synthesis of Signature Peptide for MRJP1. The selection of signature peptides is very important 1272


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Figure 2. Mass spectra of three candidate peptides of MRJP1 in the tryptic products of RJ samples by Q-TOF.

transitions contain stable isotope-labeled sites. In general, the C-terminal K or R due to trypsin treatment are isotopically labeled because this results in all y-ions being isotipically labeled. Similarly, C-terminal R of FFDYDFGSDER is isotopically labeled to obtain desired y-ions during the LC− MS/MS analysis. 3.4. Selection of MRM Transitions. MRM transitions were selected based on the rules proposed by Yocum and Chinnaiyan.31 First, doubly charged precursor is preferable to singly charged ion for the first quadrupole when ESI is used. Second, Y-ions are preferable to b-ions. Third, product ions should consist of not less than four amino acids. As expected, peptide FFDYDFGSDER showed higher y-ions than b-ions in MRJP1 tryptic products (Figure 3). Subsequently, the selected signature peptide FFDYDFGSDER and isotope-labeled peptide FFDYDFGSDER* (R*-13C6, 15N4) were fragmented to obtain the ion transitions (Figure 4). C-terminal y-ions of the two peptides were detected outstandingly well in the MS/MS spectra of their precursor ions. The singly charged ions 1103.4390 (y9), 825.3442 (y7), 563.2456 (y5) of signature peptide and 1112.9646 (y9), 835.0369 (y7), 573.0739 (y5) of isotope-labeled peptide were markedly higher than other yseries ions, so they were exploited for identification purpose. The identification MRM transition ion-pairs were shown in Figure S2, and these transitions confirmed the abovementioned rules31 of MRM transitions for protein absolute quantification peptide. Since y9 (m/z 1103.4390) and y7 (m/z 825.3442) ions were the two highest y-series ions in the

their corresponding singly charged form because of their high signal intensity in the tryptic RJ samples (Figure 2). Finally, the peptide FFDYDFGSDER was selected and synthesized as the signature peptide of MRJP1 due to its highest signal intensity (Figure 2), sensitivity, specificity, and the biggest probability (Table S1). Furthermore, the signature peptide was also confirmed to be absent in the undigested RJ samples by LC− MS/MS analysis. Thus, there is no endogenous interference occurred in the quantification of MRJP1 when peptide FFDYDFGSDER used as the specific peptide for MRJP1. 3.3. Selection and Synthesis of Isotopically Labeled Internal Standard Peptide for MRJP1. It is well-known that LC−MS/MS is a highly sensitive and selective tool for protein quantification; however, its accuracy is largely affected by ionization efficiency of the analytes in various matrices.22 To minimize the ionization efficiency variability, a stable isotopelabeled peptide FFDYDFGSDER* (R*, Arg-OH-13C6, 15N4) was synthesized and used as an internal standard (IS). Peptide sequence of the IS is the same as the signature peptide except for the C-terminal amino acid R labeled with 13C and 15N. The internal standard peptide with an incorporated stable isotope labeling can guarantee that the IS and corresponding endogenous peptide share the same physicochemical properties. Some studies have used such isotope-labeled peptides as internal standards to quantify sweet cherry fruit20 proteins and bovine lactoferrin.21 According to the recommended MRM transition selection rules,31 Y-ions are frequently preferable to b-ions to ensure that 1273


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Figure 3. Fragment ions and amino acid sequence of peptide FFDYDFGSDER from in-gel tryptic products of MRJP1 (excised protein band 1 in Figure 1). (a) Theoretical fragment ions (b ions and y ions) and amino acid sequence of peptide FFDYDFGSDER; (b) MS/MS spectrum of peptide FFDYDFGSDER.

Figure 4. MS/MS spectra of the doubly protonated peptides synthesized in the study. (a) Signature peptide FFDYDFGSDER, parent ion m/z 699.07 (2+); (b) isotope-labeled peptide FFDYDFGSDER*, parent ion m/z 704.06 (2+).

z 835.0369) were used as the quantification ions in the isotopelabeled peptide. The signature peptide FFDYDFGSDER and its

signature peptide, they were chosen as the quantification purpose. Correspondingly, y9 (m/z 1112.9646) and y7 ion (m/ 1274


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Figure 5. MRM quantitative ion-pairs of MRJP1 in a tryptic RJ sample. (a) Mass transitions of the signature peptide FFDYDFGSDER, m/z 699.5/ 1103.3 and 699.5/825.2. (b) Mass transitions of the internal standard peptide FFDYDFGSDER*, m/z 704.2/1113.2 and 704.2/835.2.

Table 2. Validation Parameters of the Developed Method

a

spiked concentration (ng/mL)

original level in tryptic sample (ng/mL)

determined level (ng/mL)

recovery (%)

intraday precision (RSD%, n = 9)

interday precision (RSD%, n = 9)

10 50 100

5.06 ± 0.22a 43.83 ± 2.20 98.01 ± 2.60

13.59 ± 0.71 91.11 ± 3.48 193.80 ± 4.30

85.33 ± 5.11 94.56 ± 3.80 95.80 ± 1.97

0.40 1.17 1.66

4.97 3.42 3.38

Data are shown as means ± SD, n = 3.

3.6. Determination of MRJP1 in RJ Samples. The presence of MRJP1 was suggested as a promising index for evaluation of RJ authenticity in quality and standardization of RJ.2 MRJP1 was also suggested as a marker for the freshness of RJ because of its degradation associated with storage temperature and storage time.16,18 Despite increasing research in proteomics and physics of RJ proteins,27,32,33 the quantification of RJ proteins in honeybee products is still very few.17−19 In this study, a UPLC−MS/MS method with a signature peptide and a stable isotope-labeled internal standard peptide was developed for the first time to quantify MRJP1 in the fresh RJ samples. MRJP1 was determined by the developed method in 28 RJ samples and two honey samples. Total proteins and MRJP1 content of these samples were shown in Table 3. Total protein contents in most RJ samples were between 13.65 and 15.17% (Table 3), which were within the scope of the values provided by many countries’ investigations and regulations.2 The MRJP1 level was 41.96−55.01 mg/g in the fresh RJ samples (Table 3). Yamaguchi et al. were the first to quantify MRJP1 content in commercial fresh RJ samples with specific MRJP1 antibody, and their obtained value was 41.4−47.2 mg/g,19 which was agreement with our MRJP1 content. However, the report lacked the essential information on how to design peptides specific for only MRJP1, or any detailed data for method validation. Bilikova and Simuth17 used ELISA and an anti-RMRJP1 antibody to measure MRJP1 in 25 honey samples (0.08−0.31 mg/g) and one RJ sample (3.35 mg/g), whose

corresponding isotope-labeled peptide from spiked internal standard were successfully detected in the tryptic products of all RJ samples (typical quantitative chromatogram is shown in Figure 5). 3.5. Method Validation. The internal standard method was used to calibrate the system for MRJP1 quantification. The method exhibited good linearity between the analyte/IS peptide peak area ratio versus analyte/IS concentration ratio in the range of 5−1000 ng/mL (signature peptide). The correlation coefficient (r2) of the linear regression equation was greater than 0.9998. The LOD (S/N = 3) and LOQ (S/N = 10) of MRJP1 in fresh royal jelly were found at 7.77 μg/g and 27.25 μg/g, respectively. The spiking recovery rates were carried out in three different signature peptide concentrations at low (10 ng/mL), medium (50 ng/mL), and high (100 ng/mL) levels and a fixed concentration of IS peptide (100 ng/mL), and results showed that the recovery rates were 85.33%, 94.56%, and 95.80%, respectively. Intraday and interday precisions were evaluated by analysis of spiked samples at different times on the same day and on the consecutive days at the concentration levels of 10, 50, and 100 ng/mL. For the target analyte obtained from both intra- and interday precision assays (Table 2), RSD values in the experiment were less than 1.66% and 4.97%, respectively. All the results obtained above demonstrated that the established method in our study had a good recovery and precision and was able to quantify the content of MRJP1 in RJ samples. 1275


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the RJ samples detected in our study were delivered by refrigerated transport (dry ice transport) and then stored at −20 °C in the laboratory. It has been reported that RJ frozen at −20 °C represents the best way to maintain quality.16,34,35 The frozen RJ samples thawed at room temperature and then underwent sample preparation as soon as possible to minimize the quality change. In addition, the sample preparation/ processing, including centrifugation, had little effect on the protein quality because the centrifugal temperature was low and time was very short. In the following experiment, including trypsin digestion, the addition of trypsin was sufficient, so the production of peptides was complete. All the reagents used for dissolution and dilution of peptides did not result in degradation or loss of these peptides. Therefore, the low level of MRJP1 detected in the experiment is not likely resulting from sample storage or treatment. Total protein contents of samples RJ-22, RJ-24, and RJ-28 were 9.07−10.23%, which were slightly lower than the other RJ samples (13.65−15.17%). Since content of total proteins has no big difference between the three samples and the other RJ samples in our study, in theory, content of MRJP1 between them should be close. On the contrary, the content difference is very big (content difference of MRJP1 even reached more than 10 times). The result implied that the proteins in samples of RJ-22, RJ-24, and RJ-28 were not all RJ-proteins because MRJP1 is the single special protein of RJ. The manufacturer might adulterate the pure RJ with other inexpensive food materials, which have similar protein content compared to RJ. Moreover, the ratios of MRJP1 to total proteins of the three abnormal RJ samples were 1.14−4.07%, which were much smaller than 30.56−38.6% (the ratio of the normal RJ samples, Table 3). Low amount of MRJP1 and low ratio of MRJP1 to total proteins suggested that samples of RJ-22, RJ-24, and RJ-28 might be adulterated with other food materials, such as egg white, yoghurt, corn starch slurry, water, or a mixture of them,2,36 leading to the level decreasing of the characteristic protein (MRJP1) in royal jelly. The data can also suggest that the ratio of MRJP1 to total proteins of fresh RJ might be considered as a supporting criterion for adulteration of RJ. The established UPLC−MS/MS method has been successfully applied to quantify MRJP1 in RJ samples and can be used for control of RJ quality and authenticity, especially for screening of RJ adulteration based on addition of other food proteins. Maybe in the near future, the content of MRJP1 and its ratio to total proteins in RJ can be new factors for the standardization of RJ. The next purpose of us is to use the developed method to determination of MRJP1 content in royal jelly produced in different countries and provide a large number of data references for relevant departments who want to create a new royal jelly standard. In addition to MRJP1 (MRJPs family), MRJP2, MRJP3, and MRJP5, as important and specific proteins in RJ, could be simultaneously quantified in one UPLC−MS/MS performing based on each corresponding signature peptide and stable isotope-labeled internal standard peptide. Therefore, in the future, we will attempt to develop a simultaneous quantitative method for MRJP1−MRJP5 of RJ samples using the principles described in this article.

Table 3. Total Proteins and MRJP1 Contents in Fresh RJ and Honey Samples

samples

total proteins amount (%)

RJ-1 RJ-2 RJ-3 RJ-4 RJ-5 RJ-6 RJ-7 RJ-8 RJ-9 RJ-10 RJ-11 RJ-12 RJ-13 RJ-14 RJ-15 RJ-16 RJ-17 RJ-18 RJ-19 RJ-20 RJ-21 RJ-22 RJ-23 RJ-24 RJ-25 RJ-26 RJ-27 RJ-28 Honey-1 Honey-2

14.10 14.04 14.25 14.99 14.61 14.14 14.23 14.12 15.17 14.28 13.68 14.31 14.11 14.62 14.46 14.81 13.97 14.99 14.42 14.77 14.12 9.96 14.32 10.23 13.65 14.46 14.73 9.07 2.14 2.04

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.53a 0.25 0.46 0.18 0.34 0.74 0.29 0.30 0.35 0.43 0.37 0.42 0.32 0.24 0.32 0.34 0.72 0.32 0.12 0.31 0.09 0.20 0.41 0.38 0.59 0.19 0.31 0.11 0.07 0.14

MRJP1 (mg/g)

MRJP1/ total proteins (%)

MRJP1/watersoluble proteins (%)b

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

37.38 37.72 38.60 32.04 33.77 34.08 34.82 35.61 32.75 33.20 31.56 34.66 31.64 30.56 31.63 31.87 31.60 34.95 32.13 33.62 36.19 1.16 34.93 4.07 30.74 34.50 36.04 1.14 3.36 2.47

46.72 47.15 48.25 40.05 42.22 42.60 43.53 44.51 40.94 41.50 39.45 43.32 39.55 38.21 39.54 39.84 39.49 43.69 40.17 42.02 45.24 -c 43.67 38.43 43.12 45.05 4.20 3.08

52.70 52.96 55.01 48.02 49.34 48.18 49.56 50.28 49.69 47.41 43.17 49.60 44.64 44.68 45.74 47.20 44.14 52.39 46.33 49.65 51.10 1.16 50.03 4.16 41.96 49.88 53.09 1.03 0.72 0.50

1.67 2.73 2.84 3.61 4.54 3.87 2.05 2.84 1.12 2.13 2.22 3.76 2.17 1.27 1.48 0.58 3.37 1.26 2.57 1.66 5.59 0.19 2.50 0.11 2.97 2.41 3.07 0.07 0.09 0.04

Data are shown as means ± SD, n = 3. bWater-soluble proteins were estimated to account for approximately 80% of the total proteins in the fresh RJ. c“-” means the value not determined. a

results were quite different from our research (in our study, MRJP1 in honey and fresh RJ samples were 0.50−0.72 mg/g, and 41.96−55.01 mg/g, respectively). However, the specificity of the anti-R-MRJP1 antibody designed by Bilikova was not high enough for accurately determining MRJP1 content because the quantification reflects the total amount of MRJP family members.18,29 On the contrary, the signature peptide FFDYDFGSDER selected in our study only exists in MRJP1, and its specificity and sensitivity are high enough to quantify MRJP1 in RJ. In our research, the ratio of MRJP1 to watersoluble proteins of fresh RJ was 38.21−48.25% (Table 3), and this value was consistent with the contents of MRJP1 for fresh RJ in previous reports, which showed MRJP1 constitutes about 45% of water-soluble proteins in fresh RJ.9 In this study, it is worth noting that there were three abnormal MRJP1 values (1.16, 4.16, and 1.03 mg/g) corresponding to samples of RJ-22, RJ-24, and RJ-28, respectively. RJ-22 to RJ-28 samples were all purchased from local markets with the price varied from high to low. MRJP1 content of RJ-23, RJ-25, RJ-26, and RJ-27 was between 41.96 and 53.09 mg/g, while RJ-22, RJ-24, and RJ-28 was ranged 1.03 to 4.16 mg/g. Contents of the latter were significantly less than the values (41.96−55.01 mg/g) obtained in our experiment. All

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Multiple sequences alignment of MRJP1−9 (Apis mellifera); MRM chromatograms of MRJP1 signature peptide FFDYDFGSDER and its corresponding internal standard peptide; candidate peptides information on MRJP1 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Linglin Fu: 0000-0001-5623-3047 Author Contributions

N.L. and S.C. contributed the same to the manuscript. N.L. and H.Z. proposed and designed the experiments. N.L., S.C., and J.L. participated in the experiments. N.L. analyzed the data and wrote the manuscript. All authors have revised the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key Technology Research and Development Program of China (2016YFD0401200) and also was supported by Y17C200016 National Science Foundation of Zhenjiang Province.

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Quantification of Major Royal Jelly Protein 1 in Fresh Royal Jelly by

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