Towards posttranslational modification proteome of royal jelly

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 53 2 7 –5 34 1

Available online at www.sciencedirect.com

www.elsevier.com/locate/jprot

Towards posttranslational modification proteome of royal jelly Lan Zhang, Yu Fang, Rongli Li, Mao Feng, Bin Han, Tiane Zhou, Jianke Li⁎ Key Laboratory of Pollinating Insect Biology, Ministry of Agriculture/Institute of Apicultural Research, Chinese Academy of Agricultural Science, Beijing, China

AR TIC LE I N FO

ABS TR ACT

Article history:

Royal jelly (RJ) is a secretory protein from the hypopharyngeal glands of nurse honeybee

Received 16 April 2012

workers, which contains a variety of proteins of which major royal jelly proteins (MRJPs) are

Accepted 13 June 2012

some of the most important. It plays important roles both for honeybee and human. Each

Available online 20 June 2012

family of MRJP 1–5 displays a string of modified protein spots in the RJ proteome profile, which may be caused by posttranslational modifications (PTMs) of MRJPs. However,

Keywords:

information on the RJ PTMs is still limited. Therefore, the PTM status of RJ was identified by

Royal jelly

using complementary proteome strategies of two-dimensional gel electrophoresis (2-DE),

Posttranslational modification

shotgun analysis in combination with high performance liquid chromatography-chip/

Two-dimensional

electrospray ionization quadrupole time-of-flight/tandem mass spectrometry and bioin-

gel electrophoresis

formatics. Phosphorylation was characterized in MRJP 1, MRJP 2 and apolipophorin-III-like

Shotgun

protein for the first time and a new site was localized in venom protein 2 precursor.

Tandem mass spectrometry

Methylation and deamidation were also identified in most of the MRJPs. The results indicate that methylation is the most important PTM of MRJPs that triggers the polymorphism of MRJP 1–5 in the RJ proteome. Our data provide a comprehensive catalog of several important PTMs in RJ and add valuable information towards assessing both the biological roles of these PTMs and deciphering the mechanisms underlying the beneficial effects of RJ for human health. © 2012 Elsevier B.V. All rights reserved.

1.

Introduction

Royal jelly (RJ) is a creamy white secretion from the hypopharyngeal glands of young workers [1,2]. It is the only food that queen and larvae less than 72 h old eat [1,2]. RJ contains proteins, sugars, lipids, vitamins and some free amino acids [1–3]. Proteins account for >50% of RJ dry weight [1–3]. RJ is one of the most popular healthy foods and claims various medical functions such as antibacterial [4], antioxidation [5], antitumor [6] and enhancing immune activity [7,8]. Beginning in the 1940s [9], its chemical compositions and pharmacological functions have been well documented [3,4,7,10–16]. Recently it has been reported that RJ has a complex proteome [17] and nine families of major royal jelly

proteins (MRJPs, MRJP 1–9) [18,19] are the most important components constituting 80–90% of the total protein content of RJ proteins [20,21]. The MRJPs are in groups of closely related proteins encoded by a family of genes [22–24]. Each family of MRJP 1–5 displays a string of distinct isoelectric point (pI) drifted protein spots by two-dimensional gel electrophoresis (2-DE) [25,26]. This might be caused by posttranslational modifications (PTMs) occurring in MRJPs [26]. Evidence about the type and site of PTMs in RJ proteins is still lacking. To provide comprehensive insight into protein functions, the covalent modifications of the proteins need to be identified and included in the proteomic analysis [27,28]. The direct identification of the type and position of PTMs in proteins is often an essential first step towards ascertaining its biological

⁎ Corresponding author at: Key Laboratory of Pollinating Insect Biology, Ministry of Agriculture/Institute of Apicultural Research, Chinese Academy of Agricultural Science, Beijing 100093, China. Tel./fax: +86 10 6259 1449. E-mail address: [email protected] (J. Li). 1874-3919/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2012.06.008

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roles [28]. Reversible phosphorylation in proteins is one of the most widespread and important PTMs, which mostly occurs on side chains of serine, threonine and tyrosine residues [28,29]. Also it plays the key role in many biological pathways, such as cell division, signal transduction, regulating protein localization, regulating enzyme activity and metabolic maintenance [29,30]. Furthermore, phosphorylation in secretory proteins such as casein in milk can stabilize high levels of calcium phosphate for the formation of micelles which increases the nutritional value of the proteins [31]. Phosphorylation can drift the protein pI from basic to acidic by providing negative charge to side chains of serine, threonine and tyrosine residues [28,32]. Protein methylation usually happens on oxygen atoms of the side chain carboxylates of aspartic acid and glutamic acid, or sometimes on nucleophilic side chains of amide nitrogens of asparagine, glutamine, ε-amine of lysine and imidazole ring of histidine. This modification increases the protein hydrophobicity and the glycosylation reactivity [28]. In contrast to phosphorylation, methylation can change the protein pI from acidic to basic by covering up the negative charge on the carboxylate side chain of the above-mentioned amino acids [28,32]. In addition, protein deamidation occurs on glutaminyl and asparaginyl residues, which can change protein structure and further affect the protein properties [33–35]. It is well known that the 2-DE technique has the advantage of visualizing modified proteins as multiple spots, whereas it excludes some of proteins with low abundance, extreme pI (<4 or >10) and molecular mass (Mr) (<15 or >200 kDa) [36,37]. In contrast, the shotgun strategy is largely unbiased, allowing both high and low abundance proteins and proteins with extremes in pI and Mr, to be identified with equal sensitivities [37,38]. The high resolution and sensitivity of liquid chromatography–tandem mass spectrometry (LC–MSn), shotgun analysis and 2-DE have become increasingly popular platforms to be successfully utilized in the proteomics survey of phosphorylation [39,40], methylation [32,41], and glycosylation [42–44] sites. Concerning RJ, only two phosphorylation sites are reported in venom protein 2 [25] and potential phosphorylation has been reported in MRJP 2, but specific sites are not assigned yet [45]. As mentioned above, each family of MRJP 1–5 in the RJ proteome displays a string of distinct pI shifted protein spots by 2-DE [25,26]. Also it well known that the protein phosphorylation and methylation can be visualized using the pI drift [28,32]. In addition, RJ is sensitive to temperature and degrades significantly when kept under room temperature for a short period of time [26,46,47]. It is known that deamidation in proteins is a spontaneous nonenzymatic reaction that triggers the protein degradation [35] and the rate of deamidation (number of deamidated peptides/total number of peptides) mainly depends upon temperature and protein primary sequence [33–35]. Therefore, the aim of this study is to investigate the position of phosphorylation, methylation and deamidation in RJ proteome using complementary proteomic approaches of 2-DE and shotgun analysis. This will not only add valuable information towards verifying the biological roles of these PTMs in RJ and extend our knowledge about the biochemical natures of RJ, but will also help decipher the mechanisms underlying the beneficial effects of RJ for human health.

2.

Materials and methods

2.1.

Chemical regents

Acrylamide, tris-base, ammonium persulfate (AP), sodium dodecyl sulfate (SDS), N,N,N′,N′-tetramethylethylene diamine (TEMED), glycin, urea, agarose, ammonium bicarbonate (NH4HCO3), and formic acid were purchased from Sigma. N, N′-methylenebisacrylamide, bromophenol blue, thiourea, 3[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), glycerol, Coomassie brilliant blue (CBB) G-250, and bovine serum albumin (BSA) were bought from Amersco. Immobilized pH gradient (IPG) strip (pH 3–10, linear), biolyte (pH 3–10), and mineral oil were purchased from Bio-Rad Laboratories Ltd. Dithiothreitol (DTT) and iodoacetamide (IAA) from Merk (Darmstadt, Germany). Trypsin was bought from Promega. Trifluoroacetic acid (TFA), acetone, and acetonitrile (ACN) were from J. T. Baker. TiO2 (5μm particles) was purchased from Titansphere, GL Science.

2.2.

Sample preparation

RJ was collected at 72 h after bee larvae were transferred into the queen cell and gathered as a pooled sample from 250 queen cell cups of five colonies (Apis mellifera L.) from the apiary of the Institute of Apicultural Research, Chinese Academy of Agricultural Science. Then RJ proteins were extracted immediately after the RJ sample was gathered according to our previously described methods with minor modification [26]. In brief, the fresh RJ (1 mg of RJ/10 μl of buffer) was mixed with a lysis buffer, containing 8 M urea, 2 M thiourea, 4% CHAPS, 20 mM tris-base, 30 mM DTT, and 1.25% Biolyte, pH 3–10. The mixture was homogenized for 5 min on ice, sonicated for 2 min and centrifuged twice at 15,000 g at 4 °C for 10 min. The debris was discarded and the supernatant was removed and placed into a new tube. Acetone was added to the collected supernatant for a final concentration of 80% (V/V) and the mixture was kept on ice for 30 min for protein precipitation. Subsequently, the mixture was centrifuged twice at 15,000g at 4°C for 10min. The supernatant was discarded, and the pellets were divided into two parts for the next analysis. Protein concentration was determined according to the Bradford method [48]. BSA was used as standard, and absorption was measured at 595nm with a DU800 spectrophotometer (Backman Coulter, Los Angeles, CA).

2.3.

2-DE-based analysis

2.3.1.

2-DE analysis

2-DE was performed by the previously described protocol [26]. Briefly, the first part of the above protein extraction (1 mg RJ/ 4 μl buffer) was dissolved in lysis buffer, then the mixture (1 μl mixture/4 μl buffer) was added into rehydration buffer (8 M urea, 2% CHAPS, 0.001% bromophenol blue, 45 mM DTT, and 0.5% Biolyte, pH 3–10). About 300 μg of protein dissolved in lysis buffer and rehydration buffer was loaded on the IPG strip (17 cm, pH 3–10, linear, Bio-Rad, Hercules, CA). Isoelectric focusing (IEF) was performed at 18 °C using a PROTEAN IEF Cell (Bio-Rad, Hercules, CA) according to the following program:

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50 V for 14 h; 250 V for 30 min for four times; 1000 V for 1 h; 9000 V for 5 h; and 9000 V for 60,000 V·h. Before sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the IPG strip was first equilibrated for 15 min at 18 °C in buffer I [6 M urea, 0.375 M Tris–HCl (pH 8.8), 20% glycerol, 2% SDS, 2% DTT] and in buffer II [6 M urea, 0.375 M Tris–HCl (pH 8.8), 20% glycerol, 2% SDS, 2.5% iodoacetoamide] for 15 min at 18 °C. After the equilibration, the strip was transferred to the SDS-PAGE gel (12% T separating gel, 1 mm). The SDS-PAGE was performed on a PROTEAN xi Cell (Bio-Rad, Hercules, CA) at 25 mA/gel for about 5.5 h at 18 °C. The gel was stained with CBB G-250 and scanned with an Image Scanner III (GE-081_TY0338, GE Healthcare).

2.3.2.

In-gel digestion for MS/MS

The CBB stained spot was excised and destained in 100 μl solution containing 25mM (NH4)HCO3 and 50% ACN until the gel spot was transparent. Then the colorless gel spot was dried with 100μl 100% ACN twice for 10min and then further dried for 30min using a Speed-Vac system (RVC 2–18, Marin Christ, Germany). Next, 10μl of trypsin solution (final concentration 10ng/μl) was added to the dried gel spot and incubated for 14h at 37°C. To extract the peptide fragments from the trypsin digests, 30μl extracted buffer I (5% TFA) was added and incubated at 37°C for 1 h. Then the supernatant was transferred into a new tube. Next, 30μl extracted buffer II (2.5% TFA, 50% ACN) was added to the digested gel and incubated at 30°C for 1 h. The supernatant added to and mixed with the supernatant from the previous step. Finally, the mixed supernatant was concentrated to 20μl using a Speed-Vac system (RVC 2–18, Marin Christ, Germany) for MS/MS.

2.3.3.

MS/MS analysis

The digested protein spot was analyzed by the high performance liquid chromatography-chip/electrospray ionization-quadrupole time-of-flight/tandem mass spectrometry (HPLC-Chip/ESI-QTOF/MS/MS) system equipped with an autosampler G1377D (maintained at 4°C), a capillary sample loading pump G1382A, a nano pump G2225A and an HPLC-Chip interface (Chip Cube G4240A) (Agilent Technologies, Santa Clara, CA). The HPLC-chip (phosphochip, G4240-620021, Agilent Technologies, Santa Clara, CA) consisted of a RP/TiO2/RP enrichment column and a ReproSil-Pur C18-AQ analytical column (75μm×150mm, 5μm), both with graphitized carbon (5mm) as stationary phase. All data were acquired in the positive ionization mode within the mass to charge ratio (m/z) range of 300–3000. Briefly, the injection program was performed by the following standard injection and nanoflow pump gradient. The loading flow rate was 3μl/min, the loading mobile phase was water with 0.1% formic acid. Elution from the analytical column was performed by a binary solvent mixture composed of water with 0.1% formic acid (solvent A) and ACN with 0.1% formic acid (solvent B). The following gradient program was used: from 3% to 8% solvent B for 1min, from 8% to 40% B for 5min, from 40% to 85% B for 1min, and 85% B for 1min. The chip flow rate was 500nl/min. The MS/ MS conditions were performed as follows: Vcap at 1900V, drying gas flow rate at 2.5L/min, drying gas temperature at 300°C, fragmentor voltage at 150V, skimmer voltage at 65V, reference masses at m/z 149.02332 and 1221.02332, max. precursor per cycle at three. About 10μl of sample was injected. At this step,

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only unphosphorylated peptides were analyzed while phosphorylated peptides were retained on the TiO2 enrichment column. After the injection program, once 15 μl elution buffer was injected (Phosphochip Kit, Agilent Technologies, Santa Clara, CA), bound phosphopeptides were released from the TiO2 column and analyzed by another regular reverse phase gradient. The loading flow rate was 3 μl/min, the loading mobile phase was water with 0.1% formic acid. Elution from the analytical column was performed by a binary solvent mixture composed of water with 0.1% formic acid (solvent A) and ACN with 0.1% formic acid (solvent B). The following gradient program was used: from 3% to 45% solvent B in 30 min, from 45% to 85% B in 3 min, and 85% B for 1 min. The chip flow rate was 300 nl/min. The MS/MS conditions were the same as the above injection program except for max. precursor per cycle set to six. Finally, a short regeneration run followed in order to maintain specificity of the phosphopeptide enrichment column. More than 4 μl of regeneration solution (Phosphochip Kit, Agilent Technologies, Santa Clara, CA) was injected onto the enrichment column. The regeneration program parameters were the same as for the above standard injection.

2.4.

Shotgun analysis

2.4.1.

In-solution digestion

The second part of the above protein extraction was dissolved in 40 mM (NH4)HCO3 and reduced with 100 mM DTT [protein solution/DTT (V/V, 10:1)] for 1 h. The mixture was then alkylated with 100 mM iodoacetamide [DTT/iodoacetamide (V/V, 1:5)] for 1 h in the dark. Next, the protein mixture was digested by trypsin (20 ng/μl) in the ratio of 1:62.5 [enzyme/ protein (W/W)] at 37 °C for 14 h. After digestion, formic acid (1 μl) was added to the solution to stop the reaction and then concentrated by the Speed-vac system (RVC 2–18, Marin Christ, Germany) to less than 100 μl.

2.4.2.

Phosphopeptide enrichment using TiO2

Phosphopeptide enrichment was performed using TiO2 chromatography [39]. Specifically, digested peptide sample was reconstituted in 200 μl binding solution (2% TFA/65% ACN). Then, 20 μl of the prepared TiO2 slurry (10 mg/ml in binding solution) was added to the above mixture and incubated at room temperature for 60 min. The supernatant was discarded and the precipitate was washed with 200 μl binding solution, 0.5% TFA/65% ACN, and 0.1% TFA/50% ACN. Finally, the phosphopeptides were twice eluted off the precipitate with 300 μl elution solution (0.3 M NH3·H2O/50% ACN) and the fractions were combined. Subsequently it was concentrated by the Speed-vac system (RVC 2–18, Marin Christ, Germany) to 50 μl for further MS/MS analysis.

2.4.3.

MS/MS analysis

The enriched phosphopeptides were also analyzed by the HPLC-Chip/ESI-QTOF/MS/MS system. The only difference was that the phosphochip was changed for a “regular” C18 chip (G4240-62010, Agilent Technologies, Santa Clara, CA). The chip was comprised of a Zorbax 300 SB-C18 enrichment column (160 nl, 5 μm) and a Zorbax 300 SB-C18 analytical column (75 μm×43 mm, 5 μm). The loading flow rate was 4 μl/

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min, the loading mobile phase was water with 0.1% formic acid. Elution from the analytical column was performed by a binary solvent mixture composed of water with 0.1% formic acid (solvent A) and ACN with 0.1% formic acid (solvent B). The following gradient program was used: from 3% to 8% solvent B for 1 min, from 8% to 40% B for 79 min, from 40 to 85% B for 30 min, and 85% B for 2 min. The chip flow rate was 500 nl/min. The MS/MS conditions were the same as the above MS/MS conditions of 2-DE but the max. precursor per cycle was 10. Almost 10 μl of the sample was injected. All data were acquired in the positive ionization mode within a range of m/z 300–3000.

2.5.

Data analysis

Tandem mass spectra were retrieved using MassHunter software (Agilent Technologies, Santa Clara, CA). The data were stored in a combined mgf file and searched against the sequence database generated from protein sequences of A. mellifera (downloaded May, 2011) augmented with sequences from Drosophila melanogaster (downloaded May, 2011), Sacharomyces cerevisiae (downloaded May, 2011) and common repository of adventitious proteins (cRAP, from The Global Proteome Machine Organization, downloaded May, 2011) using in-house Mascot (version 2.3.2, Matrix Science Ltd.). Carbamidomethyl (C) was selected as a fixed modification. Deamidated (NQ), methyl (DE), oxidation (M), phospho (ST), and phospho (Y) were selected as variable modifications. Other parameters used were as follows: enzyme, trypsin; missed cleavages, 1; peptide tolerance, 25 ppm; MS/MS tolerance, 0.03 Da. When an identified protein was matched to multiple members of a protein family or a protein appeared under the same names and accession number, the match was made in terms of higher Mascot score and differential patterns of protein spots on 2-DE gels. Protein identification was accepted that contained at least two identified peptides with minimal probability of 95%. Furthermore, all the identified PTM peptides were manually checked and evaluated by the following cut-off criterion: Mascot expectation value≤ 0.00001, and the majority of y or b ions can be detected with continuous and strong intensity peaks (some representative MS/MS spectra of the PTMs were shown in Fig. S1).

2.6.

Verification of PTMs

To confirm the PTMs of RJ, some PTM peptides were commercially synthesized using solid phase peptide synthesis process (Beijing Scillight Biotechnology Ltd. Co.). The MS/MS spectra were compared between the digested PTM peptides from RJ proteins and the synthetic PTM peptides. The PTMs were validated when almost identical MS/MS spectra were observed between the RJ and synthetic PTM peptide.

3.

Results

3.1.

PTMs analysis by 2-DE

RJ protein spots were reproducibly displayed on 2-DE images visualized by CBB stain as previously reported [26]. A total of 32

Fig. 1 – Separation of royal jelly proteins by two-dimensional gel electrophoresis (2-DE). A 300 μg protein sample was subjected to 2-DE and the proteins were stained by Coomassie brilliant blue G-250 and 32 protein spots (labeled by red code and numbered 1–32, respectively) were excised and identified by high performance liquid chromatography-chip/ electrospray ionization-quadruple time-of-flight/tandem mass spectrometry.

spots (Fig. 1), with Mr from 49 to 80kDa and pI from 4.00 to 9.00 showing a string of multi-spots were picked for further analysis of modification status by employing a phosphochip-based HPLC-chip/ESI-QTOF/MS/MS system. These 32 spots were identified as MRJP 1–5 (Table 1). Spots 1–5 were unambiguously determined to be MRJP 1 (sequence coverage>70%), with the same theoretical Mr (49.31kDa) and pI (5.10), though their experimental pI obviously drifted (5.10–5.47) (Table 1 and Fig. 1). Spots 6–12 were determined as MRJP 2 precursor (sequence coverage~70%), with the same values of theoretical Mr (51.44kDa) and pI (6.83). Here, experimental pI (6.51–7.51) still varied (Table 1 and Fig. 1). Spots 13–19 were identified as MRJP 3 (sequence coverage~50%) showing the same theoretical Mr (61.62kDa) and drifted pI (theoretical~6.47 and experimental 6.76–8.25) (Table 1 and Fig. 1). Spots 20–23 were identified as MRJP 4 (sequence coverage~50%) displaying the same theoretical Mr (53.32kDa) and drifted pI (theoretical~5.89 and experimental 6.28–6.43) (Table 1 and Fig. 1). Spots 24–28 were detected to be MRJP 5 (sequence coverage 34–47%) with the same theoretical Mr (70.48 kDa) and drifted pI (theoretical~6.11 and experimental 6.45–6.79) (Table 1 and Fig. 1). Finally, spots 29–32 were also identified as MRJP 3 (sequence coverage~50%) showing the same theoretical Mr (61.62kDa) and drifted pI (theoretical~6.47 and experimental 6.77–7.34) (Table 1 and Fig. 1). These results reproducibly displayed the PTMs in MRJP 1–5 with high confidence.

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Table 1 – Identification of major royal jelly proteins by two-dimensional gel electrophoresis. Spot no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Experimental pI

5.47 5.35 5.21 5.1 5.37 7.51 7.21 6.98 6.81 6.69 6.6 6.51 6.76 6.87 7.1 7.35 7.63 7.92 8.25 6.28 6.33 6.38 6.43 6.45 6.52 6.60 6.68 6.79 6.77 6.88 7.09 7.34

Theoretical Mr.(kDa)

pI

49.31 49.31 49.31 49.31 49.31 51.44 51.44 51.44 51.44 51.44 51.44 51.44 61.62 61.62 61.62 61.62 61.62 61.62 61.62 53.32 53.32 53.32 53.32 70.48 70.48 70.48 70.48 70.48 61.62 61.62 61.62 61.62

5.1 5.1 5.1 5.1 5.1 6.83 6.83 6.83 6.83 6.83 6.83 6.83 6.47 6.47 6.47 6.47 6.47 6.47 6.47 5.89 5.89 5.89 5.89 6.11 6.11 6.11 6.11 6.11 6.47 6.47 6.47 6.47

Score

Matches

6276 1032 3574 9862 4431 2947 3743 6541 3865 5943 3198 3978 4656 6988 8342 5737 5361 9003 9687 1281 1406 1887 1574 2132 1996 1323 2407 2276 2072 2434 2726 3243

230 48 141 334 142 108 133 234 165 211 122 141 194 249 280 219 199 251 317 49 70 79 80 93 93 48 101 89 102 109 145 169

Unique

24 11 19 24 20 16 19 25 21 24 19 19 25 26 26 26 27 23 27 15 16 18 18 15 19 11 14 12 20 19 21 23

Sequence coverage

Accession number

88% 74% 78% 87% 71% 74% 68% 71% 69% 79% 68% 73% 51% 54% 51% 53% 54% 47% 58% 49% 48% 57% 52% 47% 47% 37% 40% 34% 47% 48% 49% 55%

gi|58585098 gi|58585099 gi|58585100 gi|58585101 gi|58585102 gi|58585108 gi|58585108 gi|58585108 gi|58585108 gi|58585108 gi|58585108 gi|58585108 sp|Q17060|MRJP3_APIME sp|Q17060|MRJP3_APIME sp|Q17060|MRJP3_APIME sp|Q17060|MRJP3_APIME sp|Q17060|MRJP3_APIME sp|Q17060|MRJP3_APIME sp|Q17060|MRJP3_APIME gi|284182838 gi|284182838 gi|284182838 gi|284182838 gi|284812514 gi|284812514 gi|284812514 gi|284812514 gi|284812514 sp|Q17060|MRJP3_APIME sp|Q17060|MRJP3_APIME sp|Q17060|MRJP3_APIME sp|Q17060|MRJP3_APIME

Protein name

MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP MRJP

1 1 1 1 1 2 2 2 2 2 2 2 3 3 3 3 3 3 3 4 4 4 4 5 5 5 5 5 3 3 3 3

precursor precursor precursor precursor precursor precursor precursor

Note: spot number corresponds to the number of a protein spot in Fig. 1. All of the identified proteins are from Apis mellifera. Accession number is the unique number given to mark the entry of a protein in the database gathered from Apis mellifera augmented with protein sequences from Drosophila melanogaster, Sacharomyces cerevisiae and common repository of adventitious proteins (cRAP, from The Global Proteome Machine Organization). Experimental pI is measured by 2-DE approach and theoretical Mr (molecular weight) and pI (isoelectric point) are identified in the Database. Score is the protein total score for the search against the database. Protein name is given when the protein was identified by high performance liquid chromatography-chip/electrospray ionization-quadruple time-of-flight/tandem mass spectrometry. Matches are the number of experimental fragmentation spectra paired to a theoretical segment of protein. Sequence coverage is the ratio of the number of amino acids in every peptide that matches with the mass spectrum divided by the total number of amino acids in the protein sequence.

The characterized type and position of PTMs in MRJP 1–5 by the phosphochip-based HPLC-chip/ESI-QTOF/MS/MS system and bioinformatics is shown in Table 2 and Fig. S2. Obviously, phosphorylated sites were observed in MRJP 1 (at S259 in spot 2, at T262 in spot 3, at T262, S272, T273 in spot 4, Table 2 and Fig. S2) and MRJP2 (at T412 in spot 6, at S284, T412 in spot 7, at S323, T412 in spot 8, at S284, T412 in spot 9, at S323, T412 in spot 10, at S284, T412 in spot 11, and at S284 and T412 in spot 12, Table 2 and Fig. S2), respectively. However, phosphorylation was not found in MRJP 3–5. At the same time, potential methylation of MRJPs showed that almost all of the spots were methylated at aspartic acid (D) or glutamic acid (E) (Table 2 and Fig. S2). In spots 1–5 (MRJP 1) 11, 3, 8, 9, and 6 methylated sites were observed respectively (Table 2 and Fig. S2). In spots 6–12 (MRJP 2) 2, 4, 5, 5, 3, 3, and 3 methylation sites were observed respectively (Table 2 and Fig. S2). Spots 14–19 (MRJP 3) had 2, 2, 3, 2, 5, and 5 methylation sites respectively though none were observed in spot 13 (Table 2 and Fig. S2).

Moreover, spots 21–23 (MRJP 4) had 1, 2 and 2 methylation sites, respectively, whereas spot 20 (MRJP 4) had none (Table 2 and Fig. S2). No methylation occurred in spot 24 (MRJP 5), but spots 25–28 had 1, 2, 3 and 3 methylation sites respectively (Table 2 and Fig. S2). Meanwhile, spots 29–32 (MRJP 3) had 0, 1, 2, and 3 methylation sites respectively (Table 2 and Fig. S2). Furthermore, to verify the methylation modification of MRJPs, YFDYDFGSEEMet R was synthesized (Beijing Scillight Biotechnology Ltd. Co.). When the MS/MS spectra were compared between the digested peptide from spot 7 (MRJP 2) (Fig. 2–1) and the synthetic peptide (Fig. 2–2), almost identical MS/MS spectra were observed. This confirms that the methylation modification indeed occurs at E49 in spot 7 (MRJP 2). Deamidation was found in most of the MRJPs (Table 2 and Fig. S2). Except for all the MRJP 3 (spots 13–19 and 29–32) all of the other MRJPs had deamidation rates (number deamidated peptides/total number of peptides) greater than 25% (Fig. 3).

Modified peptides and sites

Site*

Expect

1

R.LTSNdeaTFDYDPK.F K.YDmetDCSGIVSASK.L K.YDDmetCSGIVSASK.L R.YNdeaGVPSSLNVISK.K K.FFDYDFGSDEmetR.R R.CENPDNdeaDRTPFK.I R.RQdeaDAILSGEYDYK.N R.IMNANdeaVNELILNTR.C K.NNdeaYPSDIDQWHDK.I K.MVNNdeaDFNFDDVNFR.I K.MVNNDmetFNdeaFDDVNFR.I R.TVAQdeaSDETLQMIASMK.I R.TVAQSDETLQdeaMIASMK.I K.KVGDGGPLLQdeaPYPDWSFAK.Y K.MQKMVNNdeaDmetFNFDDVNFR.I R.LSSLAVQSLDmetCNTNSDTMVYIADEK.G R.TSDYQQNDIHYEGVQNdeaILDTQSSAK.V R.TSDYQQNdeaDIHYEGVQNILDTQSSAK.V R.TSDYQQdeaNDIHYEGVQNILDTQSSAK.V R.TSDYQQNDIHYEmetGVQNdeaILDTQSSAK.V R.TSDmetYQQNdeaDIHYEGVQNILDTQSSAK.V R.TSDYQQNDmetIHYEGVQNILDTQSSAK.V K.MTIDGESYTAQDmetGISGMALSPMTNdeaNdeaLYYSPVASTSLYYVNTEQFR.T K.MTIDmetGEmetSYTAQDGISGMALSPMTNdeaNdeaLYYSPVASTSLYYVNTEQFR.T K.MVNNdeaDFNFDDVNFR.I K.MVNdeaNdeaDFNFDDVNFR.I R.TSDYQQdeaNDIHYEGVQNdeaILDTQSSAK.V K.MTIDGESYTAQdeaDmetGISGMALSPMTNNLYYSPVASTSLYYVNTEQFR.T K.MTIDmetGEmetSYTAQDGISGMALSPMTNdeaNLYYSPVASTSLYYVNTEQFR.T K.MTIDGESYTAQDGISGMALSpPMTNdeaNdeaLYYSPVASTSLYYVNTEQFR.T K.MVNdeaNDFNFDDVNFR.I

226–236 115–126 115–126 83–95 39–49 416–427 50–62 402–415 63–75 388–401 388–401 343–358 343–358 96–114 386–401 185–209 285–309 285–309 285–309 285–309 285–309 285–309 240–284 240–284 388–401 388–401 285–309 240–284 240–284 240–284 388–401

1.40E−06 1.20E−05 1.20E−05 5.60E−08 2.30E−06 4.30E−05 4.90E−06 7.20E−06 3.50E−07 1.00E−08 5.30E−09 1.30E−14 5.60E−12 5.70E−07 1.80E−08 8.70E−08 5.10E−08 9.10E−11 9.20E−09 3.50E−07 4.40E−06 1.60E−07 6.90E−07 3.40E−08 4.50E−06 5.30E−07 6.20E−06 9.60E−08 1.50E−06 2.70E−07 4.60E−08

77 51 61 75 67 58 72 76 82 103 115 162 129 92 91 105 108 134 112 80 65 76 80 108 79 88 87 99 81 89 102

K.MVNNdeaDFNFDDVNFR.I K.MVNNDmetFNdeaFDDVNFR.I R.TVAQdeaSDETLQMIASMK.I R.TVAQSDETLQdeaMIASMK.I R.LSSLAVQSLDmetCNTNSDTMVYIADEK.G R.TSDYQQdeaNDIHYEGVQNILDTQSSAK.V R.TSDYQQNdeaDIHYEGVQNdeaILDTQSSAK.V R.TSDYQQNDmetIHYEGVQNILDTQSSAK.V R.TSDmetYQQNDIHYEGVQNILDTQSSAK.V K.MTIDGESYTAQdeaDGISGMALSPMTNNdeaLYYSPVASTSLYYVNTEQFR.T K.MTIDGESYTAQDmetGISGMALSPMTNNLYYSPVASTSLYYVNTEQFR.T K.MTIDGESYTAQDGISGMALSPMTNNLYYSPVASTSLYYVNTEmetQFR.T K.MTIDGESYTAQDmetGISGMALSPMTNNLYYSPVASTSLYYVNTEQFR.T K.MTIDGEmetSYTAQDmetGISGMALSPMTNNLYYSPVASTSLYYVNTEQFR.T K.MTIDmetGESYTAQDGISGMALSPMTNNLYYSPVASTSLYYVNTEQFR.T K.MTIDGESYTAQDGISGMALSPMTpNNdeaLYYSPVASTSLYYVNTEQFR.T R.LTSNdeaTFDYDPK.F K.YDDmetCSGIVSASK.L

388–401 388–401 343–358 343–358 185–209 285–309 285–309 285–309 285–309 240–284 240–284 240–284 240–284 240–284 240–284 240–284 226–236 125–136

2.70E−06 1.70E−07 3.70E−12 1.60E−12 3.70E−08 3.40E−07 1.30E−08 1.20E−06 2.50E−08 3.60E−07 3.20E−06 1.50E−07 3.60E−07 2.60E−08 5.60E−09 6.60E−09 7.10E−07 4.20E−06

85 79 123 140 81 104 111 79 102 92 85 93 72 81 92 102 79 79

2

3

4

Score Spot no.

Site*

Expect

Score

R.QNLEMVAQNdeaDR.T K.NYPFDVDQdeaWR.D R.QAAIQdeaSGEYDHTK.N R.YDGVPSTLNdeaVISGK.T K.GDALIVYQdeaNADDSFHR.L K.SQFGENNdeaVQYQGSEDILNTQSLAK.A K.SQFGENNVQdeaYQGSEDILNTQSLAK.A K.SQFGEmetNNVQYQdeaGSEDILNTQSLAK.A K.NGVLFVGLVGNdeaSpAVGCWNEmetHQSLQR.Q R.NTHCVNNNQNDNIQNTNNQdeaNDNNQK.N R.NTpHCVNNNQNDmetNIQNTNNQNDNNQK.N R.LTSNdeaTFDYDPR.Y

337–347 65–74 52–64 84–97 208–223 284–307 284–307 284–307 312–336 411–435 411–435 224–234

8.70E−08 9.80E−07 1.40E−06 1.50E−06 2.60E−07 3.10E−09 7.60E−14 8.80E−06 3.40E−07 2.20E−10 2.30E−07 1.20E−05

97 71 69 71 87 113 143 68 84 112 83 71

13

R.QAAIQdeaSGEYDHTK.N R.QdeaAAIQSGEYDHTK.N R.YDGVPSTLNdeaVISGK.T K.IVNdeaDDFNFDDVNFR.I K.IVNDDFNFDDVNdeaFR.I K.IVNDDFNdeaFDDVNFR.I K.SQFGENNVQdeaYQdeaGSEDILNTQSLAK.A K.SpQFGEmetNNVQYQGSEDILNTQSLAK.A K.NGVLFVGLVGNSAVGCWNEmetHQSLQdeaR.Q R.NTHCVNNNQNdeaDNIQNTNNQNDNNQK.N R.NTpHCVNNNQNDmetNIQNTNNdeaQNDNNQK.N R.QNdeaLEMVAQNDR.T R.LTSNdeaTFDYDPR.Y R.QAAIQdeaSGEYDHTK.N R.QdeaAAIQSGEYDHTK.N R.YDGVPSTLNdeaVISGK.T K.IVNdeaDDFNFDDVNFR.I K.IVNDDFNdeaFDDVNFR.I K.SQFGENNdeaVQYQGSEDILNTQSLAK.A K.SpQFGEmetNNVQYQGSEDILNTQSLAK.A K.NGVLFVGLVGNSAVGCWNEHQSLQdeaR.Q K.NdeaGVLFVGLVGNSAVGCWNEmetHQSLQR.Q R.NTHCVNNNQNDNIQdeaNTNNQNDNNQK.N R.NTpHCVNNNQNDmetNIQNTNNQNDNNQK.N K.NdeaGVLFLGLVGNSGIACVNEHQVLQR.E

52–64 52–64 84–97 386–399 386–399 386–399 284–307 284–307 312–336 411–435 411–435 337–347 224–234 52–64 52–64 84–97 386–399 386–399 284–307 284–307 312–336 312–336 411–435 411–435 317–341

2.20E−05 4.60E−06 1.90E−06 6.30E−08 1.20E−09 1.40E−10 3.70E−09 2.20E−07 4.90E−07 5.40E−09 1.70E−08 2.80E−06 2.20E−05 7.30E−06 4.20E−06 2.40E−07 4.30E−09 1.80E−08 1.80E−14 3.20E−07 2.70E−06 2.50E−07 8.60E−08 2.30E−07 1.20E−05

65 75 71 103 101 108 94 65 87 94 91 76 71 61 67 88 94 81 151 89 79 75 89 75 61

14

K.IMEmetNLPQSGR.I

362–371 2.50E−06

92

15

R.KSANdeaNLAHSMK.V K.YEmetDCSGIVSAFK.I R.NNdeaGVPSSLNVVTNK.K K.SANdeaNLAHSMK.V

28–38 121–132 89–102 28–38

1.20E−06 1.20E−05 1.60E−07 5.20E−07

65 62 75 69

16

K.IMEmetNLPQSGR.I R.TNTMVYIADEmetK.G K.NdeaGVLFLGLVGNSGIACVNEHQVLQR.E K.SGEmetFDHTK.N

362–371 202–212 317–341 62–69

1.50E−05 3.90E−06 2.80E−06 1.10E−05

76 64 65 65

10

11

12

Modified peptides and sites

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 53 2 7 –53 4 1

Spot no.

5332

Table 2 – Identification of modified peptides and sites in major royal jelly proteins by two-dimensional gel electrophoresis.

3.40E−09 8.30E−09 2.20E−07 6.70E−09 1.60E−08 1.30E−08 5.30E−08 5.20E−07 1.40E−08 7.40E−08 3.80E−08 3.50E−12 1.20E−14 1.70E−12 4.30E−08 7.50E−10 1.40E−08 1.60E−11 3.30E−09 1.50E−10 6.40E−08 1.60E−07 6.40E−06 2.10E−07 8.50E−05

91 94 91 103 106 98 89 86 103 102 105 132 153 137 108 94 91 141 119 128 88 81 82 93 64

6

R.LTSNdeaTFDYDPK.F K.YDmetDCSGIVSASK.L K.YDDmetCSGIVSASK.L R.YNdeaGVPSSLNVISK.K K.FFDYDFGSDEmetR.R R.RQdeaDAILSGEYDYK.N R.IMNANVNdeaELILNTR.C R.IMNANVNELILNdeaTR.C K.NdeaNYPSDIDQWHDK.I K.NNYPSDIDQdeaWHDK.I K.NNdeaYPSDIDQWHDK.I K.MVNNdeaDFNFDDVNFR.I K.MVNNDFNdeaFDDVNFR.I K.MVNdeaNdeaDFNFDDVNFR.I R.TVAQdeaSDmetETLQMIASMK.I K.KVGDGGPLLQdeaPYPDWSFAK.Y R.TSDYQQNDIHYEGVQNdeaILDTQSSAK.V R.TSDYQQdeaNDIHYEGVQNILDTQSSAK.V R.TSDmetYQQNDIHYEGVQNdeaILDTQSSAK.V R.TSDYQQNDmetIHYEGVQNILDTQSSAK.V R.QNLEMVAQdeaNDR.T

226–236 115–126 115–126 83–95 39–49 50–62 402–415 402–415 63–75 63–75 63–75 388–401 388–401 388–401 343–358 95–114 285–309 285–309 285–309 285–309 337–347

1.30E−06 3.40E−06 9.60E−07 4.10E−06 5.20E−07 6.50E−07 3.70E−06 1.80E−07 2.40E−08 8.20E−07 1.70E−07 1.30E−08 1.40E−08 7.40E−07 4.30E−07 5.50E−07 1.30E−08 7.30E−08 6.40E−08 6.40E−08 1.50E−06

62 77 71 64 89 83 84 76 89 86 88 109 104 93 86 91 117 105 101 97 95

R.LTSNdeaTFDYDPR.Y R.QdeaAAIQSGEYDHTK.N R.YDGVPSTLNdeaVISGK.T K.IVNdeaDDFNFDDVNFR.I K.IVNDDFNdeaFDDVNFR.I

224–234 52–64 84–97 386–399 386–399

5.50E−07 7.50E−05 4.30E−07 8.80E−10 5.40E−08

87 55 79 106 82

17

K.IMEmetNLPQSGR.I K.YEmetDCSGIVSAFK.I K.IMEmetNLPQSGR.I

362–371 3.10E−05 121–132 2.10E−06 362–371 2.70E−06

59 69 68

18

R.KSANdeaNLAHSMK.V K.NGVLFLGLVGNSGIACVNEmetHQVLQR.E K.SGEmetFDHTK.N

28–38 3.90E−09 317–341 1.30E−07 62–69 1.20E−06

85 81 62

19

K.IMENdeaLPQSGR.I K.IMEmetNLPQSGR.I R.KSANdeaNLAHSMK.V R.TNTMVYIADEmetK.G K.YEmetDCSGIVSAFK.I K.NGVLFLGLVGNSGIACVNEmetHQVLQR.E K.SGEmetFDHTK.N

362–371 362–371 28–38 202–212 121–132 316–341 62–69

1.20E−05 3.60E−07 5.30E−09 4.80E−06 1.60E−08 5.60E−08 2.50E−06

56 74 94 73 101 98 71

20

K.IMEmetNLPQSGR.I R.TNTMVYIADEmetK.G K.YEmetDCSGIVSAFK.I R.NNdeaGVPSSLNVVTNK.K K.NGVLFLGLVGNdeaSGIACVNEmetHQVLQR.E K.IKQNVPQdeaSGR.V

362–371 202–212 121–132 89–102 316–341 357–366

4.30E−06 3.10E−06 1.20E−06 2.50E−09 9.10E−08 9.10E−05

75 73 69 81 80 72

21

R.QdeaAAIQSGEYDR.T K.MSNdeaQdeaQENLTLK.E R.LSSHTLNdeaHNSDK.M R.QdeaAAIQSGEYDR.T

56–66 236–246 224–235 56–66

1.90E−06 5.50E−05 4.10E−06 1.20E−06

74 67 75 71

22

K.MSNdeaQdeaQENLTLK.E 236–246 3.50E−07 R.CANFDNQDNNHYNdeaHNHNQAR.H 414–433 2.60E−07 K.VYGMALSPVTHNLYYNSPSSEmetNdeaLYYVNTESLMK.S 252–284 6.10E−06 met R.QAAIQSGE YDR.T 56–66 4.40E−06

81 75 74 71

23

K.MSNdeaQQdeaENLTLK.E R.LSSHTLNdeaHNSDK.M K.YEmetDCSGIVSAHK.I R.CANdeaFDNdeaQDNNHYNHNHNQAR.H R.QAAIQSGEmetYDR.T

236–246 224–235 120–131 414–433 56–66

1.20E−08 1.60E−06 3.90E−08 3.30E−07 1.60E−06

102 84 81 79 75

24

K.MSNdeaQdeaQENLTLK.E R.LSSHTLNdeaHNSDK.M K.SENQGNdeaDVQYER.V K.VYGMALSPVTHNLYYNdeaSPSSEmetNLYYVNTESLMK.S K.MMHLPQdeaSNK.M

236–246 224–245 285–296 252–284 360–368

1.30E−07 2.80E−06 3.60E−05 9.20E−08 8.40E−07

77 78 61 91 76

25

R.QdeaAAIQSGEYDHTK.N R.QAAIQdeaSGEYDHTK.N R.ILGANVNdeaDLIMNTR.C K.FINNdeaDYNdeaFNEVNFR.I R.LTSNdeaTFDYDPK.Y

55–67 55–67 569–582 555–568 227–237

1.30E−06 1.50E−06 6.20E−08 9.20E−08 4.20E−06

77 76 86 89 76

55–65 55–65

2.10E−06 1.20E−06

79 71

R.QAAIQSGEmetYDH.T R.QAAIQdeaSGEYDH.T

(continued on next page)

5333

83–95 154–166 50–62 402–415 402–415 63–75 63–75 63–75 388–401 388–401 388–401 343–358 343–358 97–124 135–153 314–334 185–209 285–309 285–309 285–309 240–284 240–284 240–284 240–284 376–384

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 53 2 7 –5 34 1

5

R.YNdeaGVPSSLNVISK.K K.LLTFDLTTSQdeaLLK.Q R.RQdeaDAILSGEYDYK.N R.IMNdeaANVNELILNTR.C R.IMNANdeaVNELILNTR.C K.NdeaNYPSDIDQWHDK.I K.NNYPSDIDQdeaWHDK.I K.NNYPSDmetIDQWHDmetK.I K.MVNNdeaDFNFDDVNFR.I K.MVNNdeaDFNFDDVNdeaFR.I K.MVNdeaNDmetFNFDDVNFR.I R.TVAQSDETLQdeaMIASMK.I R.TVAQdeaSDETLQdeaMIASMK.I K.VGDGGPLLQdeaPYPDWSFAK.Y R.LWVLDSGLVNdeaNTQPMCSPK.L K.SGVLFFGLVGDmetSALGCWNdeaEHR.T R.LSSLAVQSLDmetCNTNdeaSDTMVYIADEK.G R.TSDYQQdeaNDIHYEGVQNILDTQSSAK.V R.TSDYQQNdeaDIHYEGVQNdeaILDTQSSAK.V R.TSDYQQdeaNDIHYEGVQdeaNILDTQSSAK.V K.MTIDGEmetSYTAQDmetGISGMALSPMTNdeaNLYYSPVASTSLYYVNTEQFR.T K.MTIDmetGESYTAQdeaDGISGMALSPMTNNLYYSPVASTSLYYVNTEQFR.T K.MTIDGESYTAQDGISGMALSPMTpNdeaNLYYSPVASTSLYYVNTEQFR.T K.MTIDGESYTAQDGISGMALSPMTNdeaNdeaLYYSPVASpTpSLYYVNTEQFR.T R.EYILVLSNdeaK.M

Spot no.

7

9

K.SQFGEmetNNdeaVQYQGSEDILNTQSLAK.A R.NTHCVNNNQNDNIQNTNNQND NdeaNQK.N R.NTpHCVNNNQNDmetNdeaIQNTNNQNDNNQK.N R.QNLEMVAQNdeaDR.T R.QNdeaLEMVAQNDR.T R.LTSNdeaTFDYDPR.Y K.NYPFDVDQdeaWR.D K.YFDYDFGSEEmetR.R R.QdeaAAIQSGEmetYDHTK.N K.IVNdeaDDFNFDDVNFR.I K.GDALIVYQdeaNADDSFHR.L K.SQFGENNVQdeaYQdeaGSEDILNTQSLAK.A K.SpQFGEmetNNVQYQGSEDILNTQSLAK.A R.NTHCVNNNQNDNIQNTNNQNDNdeaNQK.N R.NTpHCVNNNQNDmetNdeaIQNTNNQNDNNQK.N K.SLNdeaVIHEWK.Y R.QNdeaLEMVAQNDR.T R.QNLEmetMVAQNdeaDR.T R.LTSNdeaTFDYDPR.Y K.NYPFDVDQdeaWR.D K.YFDYDFGSEmetER.R R.QdeaAAIQSGEYDHTK.N R.QAAIQdeaSGEYDHTK.N K.IVNdeaDDFNFDDVNFR.I K.GDALIVYQdeaNADDSFHR.L K.GDALIVYQNdeaADDSFHR.L K.SQFGENNVQYQdeaGSEDILNTQSLAK.A K.SQFGENNVQdeaYQGSEDILNTQSLAK.A K.SQFGEmetNNVQYQGSEDILNTQSLAK.A K.NdeaGVLFVGLVGNdeaSAVGCWNEHQSLQR.Q K.NGVLFVGLVGNSpAVGCWNEmetHQSLQR.Q R.NTHCVNNNQdeaNDNIQNTNNQNDNdeaNQK.N R.NTpHCVNNNQNDmetNIQNTNNQNDNNQK.N R.QdeaNLEmetMVAQNDR.T R.LTSNdeaTFDYDPR.Y K.NYPFDVDQdeaWR.D K.YFDYDFGSEEmetR.R R.QdeaAAIQSGEYDHTK.N R.QAAIQdeaSGEYDHTK.N K.IVNDDFNdeaFDDVNFR.I K.GDALIVYQdeaNADDSFHR.L K.SQFGENNVQdeaYQGSEDILNTQSLAK.A K.SQFGENNVQYQdeaGSEDILNTQSLAK.A K.SQFGENNVQYQGSEmetDILNTQSLAK.A K.SpQFGEmetNNVQYQGSEDILNTQSLAK.A R.NTHCVNNNQNdeaDNIQNTNNQNDNNQK.N R.NTpHCVNNNQNDmetNIQdeaNTNNQNDNNQK.N

Site*

Expect

284–307 411–435 411–435 337–347 337–347 124–134 65–74 40–50 52–64 386–399 208–223 284–307 284–307 411–435 411–435 30–39 337–347 337–347 224–234 65–74 40–50 52–64 52–64 386–399 208–223 208–223 284–307 284–307 284–307 312–336 312–336 411–435 411–435 337–347 224–234 65–74 40–50 52–64 52–64 386–399 208–223 284–307 284–307 284–307 284–307 411–435 411–435

1.10E−10 2.60E−06 7.20E−08 6.10E−05 8.10E−05 3.70E−06 7.30E−05 2.60E−05 1.40E−06 1.60E−09 3.30E−07 1.50E−09 4.60E−07 5.10E−10 1.60E−06 2.50E−05 3.60E−05 5.50E−05 5.70E−06 8.20E−07 8.50E−06 1.50E−07 5.20E−08 7.60E−09 1.40E−08 7.20E−07 1.40E−11 8.30E−11 7.40E−08 1.10E−07 1.30E−06 2.20E−10 5.20E−07 3.30E−05 9.10E−06 8.80E−07 3.30E−06 1.20E−06 1.60E−06 5.30E−10 9.10E−08 1.20E−12 1.60E−09 2.10E−07 3.60E−06 1.30E−09 2.60E−07

Score Spot no. 105 73 89 64 67 78 53 66 74 91 89 102 91 110 72 67 65 69 68 79 76 77 89 90 104 79 107 115 87 89 73 114 89 61 65 62 79 78 75 111 93 135 96 87 76 101 88

Site*

Expect

Score

R.ILGANVNdeaDLIMNTR.C K.FINNdeaDYNdeaFNEVNFR.I R.LTSNdeaTFDYDPK.Y R.QdeaMNEmetYMMALSMK.L R.ILGANVNdeaDLIMNTR.C K.FINNDYNdeaFNEVNFR.I R.ENMDMVAQdeaNEmetETLQTVVAMK.M K.SEYGANNVQYQdeaGVQDIFNTESIAK.I R.LTSNdeaTFDYDPK.Y

569–582 555–568 227–237 540–551 569–582 555–568 340–359 287–310 227–237

1.80E−05 6.80E−07 1.60E−05 3.30E−06 2.10E−05 1.10E−06 3.80E−07 2.30E−07 4.40E−06

61 78 74 79 64 77 86 97 66

30

R.QAAIQdeaSGEYDH.T R.ILGANVNdeaDLIMNTR.C K.FINNdeaDYNdeaFNEVNFR.I R.ENMDmetMVAQdeaNEETLQTVVAMK.M R.ENMDMVAQNEmetETLQTVVAMK.M K.SEmetYGANNVQdeaYQGVQDIFNTESIAK.I R.LTSNdeaTFDYDPK.Y K.YLDmetYDFGSDER.R R.QAAIQdeaSGEYDH.T R.ILGANVNdeaDLIMNTR.C K.FINNDYNdeaFNEVNFR.I R.ENMDmetMVAQdeaNEETLQTVVAMK.M R.EmetNMDMVAQdeaNEETLQTVVAMK.M K.SEYGANdeaNVQYQGVQDIFNTESIAK.I K.SEYGANNVQdeaYQGVQDIFNTESIAK.I K.YEDmetCSGIVSAFK.I

55–65 569–582 555–568 340–359 340–359 287–310 227–237 43–53 55–65 569–582 555–568 340–359 340–359 287–310 287–310 121–132

1.80E−07 1.30E−05 6.20E−07 6.10E−07 8.30E−08 8.10E−08 9.40E−05 3.20E−06 8.10E−06 6.30E−08 8.40E−08 5.10E−06 1.50E−06 1.10E−14 3.20E−11 3.1E−07

69 63 78 93 89 97 62 69 61 93 95 84 88 156 117 83

31

K.YEDmetCSGIVSAFK.I

121–132 7.2E−08

91

32

R.INDPEGNEmetYMLALSNR.M K.YEDmetCSGIVSAFK.I

372–387 6.9E−09 121–132 1.6E−08

96 79

R.INDPEGNEmetYMLALSNR.M K.HIDFDFGSDEmetR.R

372–387 6.6E−07 45–56 3.9E−08

70 74

26

27

28

Modified peptides and sites

Note: Spot number corresponds to the number of a protein spot in Fig. 1. Site* is the position of the initial and final amino acids of the peptide in the protein sequence. Score is for the identified peptides searched against the database gathered from Apis mellifera augmented with protein sequences from Drosophila melanogaster, Sacharomyces cerevisiae and common repository of adventitious proteins (cRAP, from The Global Proteome Machine Organization). Expect value is the theoretical probability of obtaining false-positive peptide identification. Phosphorylated amino acid is labeled with Sp or Tp, methylated amino acid is marked with DMet or EMet, and deamidated amino acid is labeled with NDea or QDea, respectively.

J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 53 2 7 –53 4 1

8

Modified peptides and sites

5334

Table 2 (continued)

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 53 2 7 –5 34 1

5335

Fig. 2 – Tandem mass spectra of methylation peptide of royal jelly (RJ). Where (1) is tandem mass spectra of the methylation peptide YFDYDFGSEEmetR obtained from RJ by two-dimensional gel electrophoresis, and (2) is tandem mass spectra of the synthetic methylation peptide YFDYDFGSEEmetR. The spectra are labeled with different color codes, where the blue, green and red represent ions of a, b and y, respectively. The gray is precursor ion and marked as [M].

To provide an estimate of the degree of modification at the reported sites, the ratio of non-modified peptide numbers to modified peptide numbers was almost 1:2 or 1:3 (Table S1).

3.2.

PTM analyzed by shotgun approach

To have more complete coverage of the PTMs of RJ proteome, the shotgun technique was also used since proteins with high or low abundance and extreme pI and Mr can be comprehensively analyzed. A total of 16 protein species were determined by this approach (Table 3). Besides MRJP 1–5, 11 other proteins were identified, i.e. MRJP 6, MRJP 7, MRJP 9, glucose oxidase, glucocerebrosidase, venom protein 2 precursor, apolipophorin-III-like protein, Regucalcin (RC) (Senescence marker protein 30)

(SMP-30), alpha-glucosidase, defensin precursor, and glucose dehydrogenase isoform 1. Peptides were phosphorylated at S43, S202, S205 in venom protein 2 precursor (Table 4 and Fig. S3), and at S117, S118 in apolipophorin-III-like protein (Table 4 and Fig. S3) respectively. Phosphorylation was not detected in the other proteins. To confirm the phosphorylated modifications, the MS/MS spectra were compared between the digested peptide from the venom protein 2 precursor (Fig. 4‐1) and apolipophorin-III-like protein (Fig. 4‐3) and the synthetic peptides NVDTVLVLPSpIER (Fig. 4‐2) and VVDDISpSGIPNAQQQVAELQSK (Fig. 4‐4) (Beijing Scillight Biotechnology Ltd. Co.), respectively. Almost identical MS/MS spectra were observed between the above respective digested peptides from RJ and synthetic peptides. This

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Fig. 3 – The rate of deamidation (number deamidated peptides/total number of peptides) of major royal jelly proteins (MRJPs) by two-dimensional gel electrophoresis. Spot number corresponds to the number of a protein spot in Fig. 1. verifies that phosphorylation modification occurs at S43 in venom protein 2 precursor and at S117 in apolipophorin-III-like protein, respectively. In addition, methylation was observed in MRJP 1, MRJP 2, MRJP 3, MRJP 7, and glucose dehydrogenase isoform 1 (Table 4 and Fig. S3). Deamidation was found in almost all of the MRJPs (except for MRJP 6 and 9) and in glucose oxidase (Table 4 and Fig. S3). However, methylation and deamidation were not found in the other RJ proteins. Similar to 2-DE approach, the modification rate at the reported sites here was also 1:2 or 1:3 except the higher rate of

phosphorylation was detected because of selective phosphopeptide enrichment (Table S2).

4.

Discussion

To gain insight into the PTMs of RJ proteome, this study comprehensively investigated phosphorylation, methylation and deamidation of RJ using gel-based 2-DE and shotgun proteomic approaches. All of the RJ samples used for this study were collected directly from our laboratory apiary to

Table 3 – Identification of royal jelly proteins by shotgun approach. Protein name

Mr. (kDa)

pI

Score

Matches

Unique

Sequence coverage

Accession no.

MRJP 1 MRJP 2 precusour MRJP 3 MRJP 4 MRJP 5 MRJP 6 MRJP 7 MRJP 9 Glucose oxidase Glucocerebrosidase Venom protein 2 precursor Apolipophorin-III-like protein Regucalcin (RC) (Senescence Marker protein 30) (SMP-30) Alpha-glucosidase Defensin precursor Glucose dehydrogenase isoform 1

49.31 51.44 61.96 53.32 70.48 50.15 50.85 48.77 68.35 58.31 24.83 21.33 10.21

5.1 6.83 6.47 5.89 6.11 5.89 4.9 8.7 6.48 5.19 4.51 5.48 8.76

2652 1321 1893 1564 1126 113 2214 114 654 651 511 572 210

106 44 78 40 28 8 67 6 17 12 15 15 5

19 12 15 19 19 4 15 3 9 9 2 3 3

78% 55% 52% 70% 56% 27% 70% 8% 32% 37% 26% 33% 80%

gi|58585098 gi|58585108 sp|Q17060|MRJP3_APIME gi|284182838 gi|284812514 gi|58585188 gi|62198227 gi|189212377 gi|58585090 gi|66511547 gi|60115688 gi|166795901 gi|110758964

65.69 11.05 69.93

5.06 6.28 5.58

181 198 122

6 3 4

6 2 4

19% 33% 20%

gi|58585164 gi|562090 gi|110756961

Note: all of the identified proteins are from Apis mellifera. Accession number is the unique number given to mark the entry of a protein in the database gathered from Apis mellifera augmented with protein sequences from Drosophila melanogaster, Sacharomyces cerevisiae and common repository of adventitious proteins (cRAP, from The Global Proteome Machine Organization). Theoretical Mr (molecular weight) and pI (isoelectric point) are identified in the database. Score is the protein total score searched against the Database. Protein name is given when protein was identified by high performance liquid chromatography-chip/electrospray ionization-quadruple time-of-flight/tandem mass spectrometry. Matches is the number of experimental fragmentation spectra paired to a theoretical segment of protein. Sequence coverage is the ratio of the number of amino acids in every peptide that matches with the mass spectrum divided by the total number of amino acids in the protein sequence.

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Table 4 – Identification of modified peptides and sites in royal jelly by shotgun approach. Protein Name MRJP 1

MRJP 2 precusour

MRJP 3

MRJP 4 MRJP 5 MRJP 7

Glucose oxidase Venom protein 2 precursor

Apolipophorin-III-like protein Glucose dehydrogenase isoform 1

Modified peptides and sites

Site*

Expect

Score

K.MVN NDFNFDDVNFR.I K.MVNNDmetFNFDDVNFR.I R.LSSLAVQSLDmetCNdeaTNdeaSDTMVYIADEK.G R.TSDYQQNDIHYEGVQdeaNILDTQSSAK.V R.LTSNdeaTFDYDPR.Y K.SQFGEmetNNVQdeaYQGSEDILNTQSLAK.A R.NTHCVNNNQNdeaDNIQdeaNTNNQNDNNQK.N R.NTHCVNNNQNDmetNIQdeaNTNNQNDNNQK.N K.IINNDFNFNdeaDVNFR.I R.LWVLDSGLVNNNQdeaPMCSPK. R.YHNQNAGNQNADmetNQNdeaADNQNANNQNADNQNANK.Q R.YHNQNAGNQNADNQNdeaADmetNQNANNQNADNQNANK.Q K.MSNdeaQQENLTLK.E K.SEYGANdeaNVQYQGVQDIFNTESIAK.I K.ILNNdeaDmetLNFNDINFR.I K.VQYNdeaGVQDVFNTQTTAK.A R.ENTDmetMVAQNEETLQMIVGMK.I R.ATGVNVLINdeaGR.R R.INdeaGFTVAQTISR.N K.NVDTVLVLPSpIER.D R.SpVESVEDFDNEIPK.N R.SVESpVEDFDNEIPK.N K.VVDDISpSGIPNAQQQVAELQSK.F K.VVDDISSpGIPNAQQQVAELQSK.F R.EmetIIVSGGAVNSPQILLLSGIGPK.E

388–401 388–401 185–209 285–309 224–234 284–309 411–435 411–435 391–404 141–159 422–454 422–454 236–246 287–309 388–401 290–307 337–356 297–307 247–258 34–50 202–215 202–215 112–133 112–133 310–332

5.30E−07 3.60E−06 4.20E−06 1.50E−06 8.00E−07 8.20E−08 4.30E−10 2.30E−07 2.50E−06 5.50E−11 1.00E−07 6.10E−06 5.10E−07 5.30E−08 3.40E−09 5.10E−08 7.10E−08 7.30E−07 1.20E−06 1.50E−07 7.30E−06 2.40E−07 2.10E−13 1.20E−09 6.10E−06

86 89 64 71 78 77 106 87 73 113 82 73 81 78 93 108 87 72 68 88 76 82 137 92 65

dea

Note: site* is the position of the initial and final amino acids of the peptide in the protein sequence. Score is the identified peptides searched against the database gathered Apis mellifera augmented with protein sequences from Drosophila melanogaster, Sacharomyces cerevisiae and common repository of adventitious proteins (cRAP, from The Global Proteome Machine Organization). Expect value is the theoretical probability of obtaining false-positive peptide identification. Phosphorylated amino acid is labeled with Sp or Tp, methylated amino acid is marked with DMet or EMet, and deamidated amino acid is labeled with NDea or QDea, respectively.

ensure sample freshness and quality. To our knowledge, the sites of RJ PTMs we report here provide vital knowledge towards ascertaining their biological roles and biochemical natures in RJ and contribute to better unraveling the mechanisms underlying the beneficial effects of RJ for honeybee biology and human health. To date, only two phosphorylation sites in venom protein 2 [25] and potential phosphorylation modification in MRJP 2 have been reported, but specific sites are not mapped yet [45]. In this study, except for identifying known phosphorylation sites at S202 and S205 in venom protein 2 precursor, a new site at S43 was successively identified in this protein by the shotgun proteome technique. All of the phosphorylation sites identified in MRJP 1, MRJP 2, and apolipophorin-III-like protein are reported here for the first time. The low number of phosphorylation sites detected in MRJP 1–2 suggests that the polymorphism of MRJPs could not be the consequence of phosphorylation modification. The function of MRJPs is used to store nutrients for the fast developing honeybee larvae and highly fertile queen [49,50] and MRJP 1 is a main modulator of the honeybee caste differentiation [15]. Phosphorylation in secretory proteins permits the stabilization of high levels of calcium phosphate, which is an important source of nutrients [28,31]. Therefore, like phosphorylated casein in milk [31], the phosphorylated MRJP 1–2 are thought to support the more efficient nutrition that is required for the high nutritional demands of the fast growing honeybee larvae and active egg-laying queen to support their high nutrition demand [2]. Venom protein 2 is a serine proteinase homolog that has defense mechanisms against intruding microorganisms and parasites in

insects [51]. Usually an enzyme presents as an inactive precursor (zymogen) and can be rapidly converted to the active form by rapid dephosphorylation of serine once stimulation occurs [52,53]. In honeybees, the larvae have weak immune system during the first 48h following eclosion (egg hatching) and are susceptible to disease infection, such as Paenibacillus larvae [54]. Larvae, however, can grow well in the normal colony because they feed on RJ exclusively, which supposedly strengthens the larva immune system [1–3]. The rapid dephosphorylation of the phosphorylated venom protein 2 precursor on serine residues may function as a regulatory mechanism to protect larvae from diseases infection [52,53]. Apolipophorin-III protein is responsible for transforming phospholipid vesicles into discoidal complexes and plays a role in promoting the innate immunity [55]. Traditionally, phosphorylation modification can enhance the regulatory processes of ubiquitination for the ever-evolving immune system within an organism [56]. Hence, it is suggested that phosphorylation modification in Apolipophorin-III-like protein in RJ plays a role in strengthening the immunity of the larvae and the queens [7,8]. Protein methylation modification plays key roles in changing the biochemical features of proteins. For example, once methylation modification occurs, it covers up a negative charge and enhances the hydrophobicity of the protein [28]. In this study, methylation occurred in most of the MRJPs suggesting that it is the main PTM form in RJ. To maintain quality, RJ should retain high viscosity and form micelles [2,57,58]. Methylation in most of MRJPs suggests that it serves a major role in the formation of high viscosity micelles in RJ by strengthening

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self-association and adsorption derived from hydrophobicity [28,59]. This could satisfy the biological nature of the honeybee queen and worker larvae, helping them to develop normally through adhering to the vertical face-down queen and horizontal opened worker cells in the colony [2]. In addition, the methylation modification can enhance the lower reactivity of asparagine carboxamido nitrogen in the N-glycosylation of proteins, i.e. the methylation modification could enhance the glycosylation of proteins [28]. As far as we know, MRJPs in RJ are a family of glycoproteins [50,60,61]. This implies that the methylation in RJ could facilitate RJ protein glycosylation. Furthermore, the different numbers of methylation sites observed in the MRJP 1–5, especially spots 13–32, indicate that

the visible polymorphism (drifted pI) of the MRJP 1–5 in 2-DE profile is likely caused by methylation modification by covering up the negative charge on the carboxylate side chain to migrate the protein pI from acidic to basic [28]. However, the obvious drift of Mr (spots 13–19 with higher Mr than spots 29–32) of MRJP 3 cannot be explained by our present protocol and requires further independent investigation. Deamidation in proteins is a spontaneous nonenzymatic reaction that happens on the glutaminyl and asparaginyl residues when proteins suffer from degradation [35]. The rate of deamidation of peptides/proteins depends upon factors including protein primary sequence and temperature [33–35]. In the present study, except for MRJP 3, the higher rate of

Fig. 4 – Tandem mass spectra of posttranslational peptides of royal jelly (RJ). Where (1) is the tandem mass spectra of the phosphopeptide NVDTVLVLPSpIER obtained from RJ by shotgun, (2) is the tandem mass spectra of the synthetic phosphopeptide NVDTVLVLPSpIER, (3) is the tandem mass spectra of the phosphopeptide VVDDISpSGIPNAQQQVAELQSK obtained from RJ by shotgun, and (4) is the tandem mass spectra of the synthetic phosphopeptide VVDDISpSGIPNAQQQVAELQSK. The spectra are labeled with different color codes, where the green and orange represent ions of b and y, respectively. The gray is the precursor ion marked as [M] and the red is the precursor ion with loss of H3PO4.

J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 53 2 7 –5 34 1

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Fig. 4 (continued).

deamidation (>25%) in all other MRJPs observed by 2-DE approach suggests that the deamidation reaction could be easily triggered by high temperature, thus reducing the quality by the degradation of the RJ proteins [26]. This is supported by the idea that the RJ is sensitive to temperature and degrades significantly when kept under room temperature for a short period of time [26]. In addition, the relative lower rate of deamidation in MRJP 3 here suggests that it functions as a storage form of process-able nitrogen that requires relatively more stability [26,49,50]. In a honeybee colony, the larva growth requires a stable temperature at 34–35 °C in the hive [2]. To ensure the fast developing larvae and egg-laying queen receive the freshest and most efficient food at such an in-hive temperature, the honeybee has an evolutionary strategy that the nurse bees feed the young larvae with RJ every 0.5–3 min and the queens at any moment to prevent the RJ protein from degrading [2]. Given this biochemical nature, to maintain quality and for better utilization for human consumption, RJ should be kept under

lower temperature conditions to inhibit deamidation reaction [26]. The estimated modification rate of the RJ at the reported sites (almost 1:2 or 1:3, non-modified:modified) provides us with the first evidence of natural modification status of RJ proteins. This helps us to better understand the biochemical features of RJ proteins. In addition, the higher rate of phosphorylation that was obtained by our present protocols demonstrates a high efficiency to enrich the phosphopeptides in the RJ. Understanding the relationship between function and exact chemical composition of a defined protein species in the context of the proteome is one of the major challenges in proteomics [62]. 100% sequence coverage is one of the major prerequisites to reach the protein species level [62]. Therefore, to add more comprehensive and valuable information towards evaluating the biological roles of these PTMs, some new methods including multiple complete digest or top-down/ middle-down approaches [62] should be adopted to increase the sequence coverage of RJ proteins in the future.

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Conclusion

Since RJ plays key roles in the honeybee community and promotes various health functions in humans, analysis of its PTM proteome contributes more biochemical knowledge for the future functional development and better utilization of this natural product. This study identified several PTMs like phosphorylation, methylation and deamidation of RJ proteins using the 2-DE and shotgun complementary proteomics strategies. Specifically, the phosphorylated MRJP 1–2 are thought to increase the nutrition efficiency for honeybees. Methylation is suggested as the major cause of MRJP polymorphism and helps maintain the high viscosity and formation of micelles in RJ. The higher rate of deamidation modification in MRJPs confirms that RJ stored under lower temperature conditions could inhibit its proteins from nonenzymatic reactions that cause degradation. These comprehensive catalogs of PTMs of RJ proteins have significantly advanced our level of understanding of how PTMs benefit honeybee biology and human health at the systemic level. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2012.06.008..

Acknowledgments We thank Dr. John Kefuss, France and Dr. Paul N Goulding, UK, for their help with the language of the manuscript. We also thank Dr. Ruixiang Su, China, for his bioinformatics help. This work is supported by the earmarked fund for Modern Agro-industry Technology Research System (CARS-45) and The National Natural Science Foundation of China (no. 30972148).

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