Comprehensive Royal Jelly (RJ) Proteomics Using ... - ACS Publications

Comprehensive Royal Jelly (RJ) Proteomics Using One- and Two-Dimensional Proteomics Platforms Reveals Novel RJ Proteins and Potential Phospho/Glycoproteins Takako Furusawa,† Randeep Rakwal,‡,§ Hyung Wook Nam,| Junko Shibato,‡ Ganesh Kumar Agrawal,‡,¶ Yu Sam Kim,| Yoko Ogawa,⊥ Yasukazu Yoshida,⊥ Yoshiaki Kouzuma,† Yoshinori Masuo,‡ and Masami Yonekura*,† Food Function Laboratory, School of Agriculture, Ibaraki University, Ami 300-0393, Japan, Health Technology Research Center (HTRC), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba West, 16-1 Onogawa, Tsukuba 305-8569, Japan, Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal, Protein Network Research Center, Yonsei University, Seoul 120-749, Korea, HTRC, AIST Kansai Center, 1-8-31 Midorigaoka, Ikeda 563-8577, Japan Received January 24, 2008

Royal jelly (RJ) is an exclusive food for queen honey bee (Apis mellifera L.) that is synthesized and secreted by young worker bees. RJ is also widely used in medical products, cosmetics, and as health foods. However, little is known about RJ functionality and the total protein components, although recent research is attempting to unravel the RJ proteome. We have embarked on a detailed investigation of the RJ proteome, using a modified protein extraction protocol and two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with tandem mass spectrometry. Simultaneously, we examined total soluble protein from RJ collected at 24, 48, and 72 h after honey bee larvae deposition twice (in two flower blooming seasons), to check differences, if any, in RJ proteome therein. Both 1- and 2-D gels stained with silver nitrate revealed similar protein profiles among these three time points. However, we observed a clear difference in two bands (ca. MW of 55 and 75 kDa) on 1-D gel between the first and the second collection of RJ. A similar difference was also observed in the 2-D gel. Except for this difference, the protein profiles were similar at the 3 time points. As the RJ from 48 (or sometimes 72) is commercially used, we selected the RJ sample at 48 h for detailed analysis with the first collection. 1-DGE identified 90 and 15 proteins from the first and second selection, respectively; in total, 47 nonredundant proteins were identified. 2-DGE identified 105 proteins comprising 14 nonredundant proteins. In total, 52 nonredundant proteins were identified in this study, and other than the major royal jelly protein family and some other previously identified proteins, 42 novel proteins were identified. Furthermore, we also report potentially post-translationally modified (phosphorylation and glycosylation) RJ proteins based on the Pro-Q diamond/emerald phosphoprotein/glycoprotein gel stains; MRJP 2p and 7p were suggested as potential phosphoproteins. The 2-DGE data were integrated to develop a 2-D gel reference map, and all data are accessible through RJ proteomics portal (http://foodfunc.agr.ibaraki.ac.jp/RJP.html). Keywords: Apis mellifera • royal jelly • proteomics analysis • gel-based approach • mass spectrometry • proteins

1. Introduction From time immemorial, honey bee (Apis mellifera L.) can be considered as one of the most familiar and essential insects * To whom correspondence should be addressed. Dr. Masami Yonekura, School of Agriculture, Ibaraki University 3-21-1, Ami-machi, Ibaraki 3000393, Japan. E-mail: [email protected]. Tel/fax: +81-29-888-8683. † Ibaraki University. ‡ Health Technology Research Center (HTRC), National Institute of Advanced Industrial Science and Technology (AIST). § Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). | Yonsei University. ¶ Present address: University of Missouri, Biochemistry Department, 204 Life Sciences Center, Columbia, MO 65211. ⊥ HTRC, AIST Kansai Center.

3194 Journal of Proteome Research 2008, 7, 3194–3229 Published on Web 06/26/2008

for human and plant life. They are critical organisms for apiculture (honey production, etc.) and pollination, and it would not be wrong to state that their benefit is immeasurable. So, the research on honey bee as an organism is gaining ground, and the recent completion of the genome sequence of honey bee is a good example on the importance of this model organism as a tool for understanding the biology of insects.1 Moreover, the honey bee is also a classical model organism for elucidation of the social system.2 We briefly describe the honey bee as it is relevant to the subject of the study, namely, royal jelly (RJ), which has become an immensely popular bee-derived health food for humans. As honey bee is a eusocial insect, approximately 30 000 bees coexist per colony, 10.1021/pr800061j CCC: $40.75

 2008 American Chemical Society

RJ Proteomics Reveals Novel RJ Proteins and Phospho/Glycoproteins and these are divided into three castes: one queen (fertile female), some 20 000-40 000 workers (sterile female), and 200-300 drones (male). They have different life cycles and each plays a unique and different role. Females, workers, and the queen arise from fertilized (diploid) eggs laid by the queen. In contrast, males arise from haploid unfertilized eggs. Diploid eggs become queens or workers depending on which cell they are laid in and whether the resulting larvae are fed RJ or also known as worker jelly.3 The RJ is secreted via the glandular cephalic system (hypopharingeal and mandibular glands) of worker bees and fed to the queen for its lifetime, and to all young honey bee larvae for three days after hatching.4 The RJ is a critical source of nutrition for the larvae and the queen. Because RJ possesses several health-promoting and pharmacological properties, RJ is also widely used as in human health-promoting medical products, cosmetics, and health foods.5 Thus, RJ has been widely studied during the past 7 decades, and has resulted in about 285 scientific publications (as of January 14, 2008) on PubMed with the keyword “royal jelly”. However, there are few studies on the mechanism of individual RJ components (proteins and metabolites) as factors for these pharmacological effects ascribed to RJ. What is the composition of RJ? The RJ has an average moisture content of 60-70%, and consists of crude protein 12-15%, total sugar 10-16%, lipids 3-6%, vitamins, salts, and amino acids. Herein lies our aim, “to examine the protein component of RJ”, which is known to consist of both water-soluble (46-89%) and -insoluble proteins, by using the proteomics technology.6–8 To do so, we have embarked on a detailed and comprehensive investigation of the RJ proteome, which has also been previously9 and recently challenged,10,11 using a modified/ improved protein extraction protocol and two complementary proteomics approaches, namely, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with tandem mass spectrometry (MS/MS). Simultaneously, we examined total soluble protein from RJ collected at 24, 48, and 72 h after honey bee larvae deposition twice (in two flower blooming seasons), and compared the protein expression profiles, a novel aspect of this study. The reason to do a time course and seasonal collection study was based on the fact that RJ from the 48 h (or sometimes 72 h) time-point is commercially used. Other than proteins previously isolated and identified proteins, we identified the largest number (210 proteins representing 52 nonredundant proteins), including 42 novel proteins, indicating not only the usefulness of the modified (and optimized) protein extraction buffer system, but also the importance of using both 1- and 2-DGE approaches in conjunction with MS. Moreover, we present novel data on the potential post-translational modifications (PTMs) in the RJ proteome by both 1- and 2-DGE. As the aim of our study was also to provide a reference database for the RJ and honey bee researchers, we have also constructed a large-format and high-resolution 2-D gel reference map. The obtained protein data (1- and 2-DGE) accessible through the RJ proteomics portal (http://foodfunc.agr.ibaraki.ac.jp/RJP.html) reveals RJ (protein) functionality for insects/ human benefit.

2. Materials and Methods 2.1. Royal Jelly Collection. European honeybees (A. mellifera L.) colonies maintained at the Kato Bihoen Honpo Co., Ltd. (Yokohama, Japan) were the source for RJ used in this study. The RJ was collected at a company farm in Moriya, Japan. We briefly describe the protocol for RJ collection. After

research articles

Figure 1. Sampling of the RJ. The RJ was collected at 24, 48, and 72 h after queen honey bee larvae were transferred into the queen cell (top, diameter 10 mm; side, height 12 mm). Two different samples of RJ were collected at two different dates during the month of May, 2007; on May 7-10 (wild cherry and dandelion, etc., were blooming in/around the farm) labeled the 1st collection (called 1-24, 1-42, 1-72 RJ, respectively), and on May 21-24 (mainly R. pseudoacacia L. was blooming) labeled the 2nd collection (called 2-24, 2-48, 2-72 RJ, respectively). The 0 h indicates the time point at which a drop of RJ was added to the artificial queen cell over which the queen bee larvae (1/2day-old) was placed. Green arrows indicate developing queen honey bee larvae in RJ.

the artificial queen cells (diameter 10 mm × 12 mm (height), and see Figure 1) were prepared, RJ (mixture of 50% freshfrozen RJ and 50% Milli Q (MQ) water) was added into the cups. The female honeybee larvae (2/3-day-old and ca. 2 mm in length) were gently placed into the cups. The RJ was collected from the cups at 24, 48, and 72 h postaddition by first removing the larvae using forceps, followed by removing the RJ with a soft spoon. The RJ collection was done twice on these dates: 2007/05/07-05/10 (wild cherry, dandelion, etc., were blooming) labeled as the first collection (these RJs are called 1-24, 1-42, 1-72 RJ, respectively), and 2007/05/21-05/24 (Robinia pseudoacacia L. was in full bloom) labeled as the second collection (these are called 2-24, 2-48, 2-72 RJ). The collected RJ was stored deep frozen at -80 °C. We greatly appreciate the close cooperation with the farm staff and the Kato Bihoen Honpo Co., Ltd., for the sampling of RJ. 2.2. Extraction of Total Protein. Each RJ (ca. 100 mg) was mixed with 3 mL of homogenization buffer [HB, 0.2 M TrisHCl buffer, pH 7.8, containing 5 mM EDTA · 2Na, 14 mM 2-ME, 10% (v/v) glycerol, and 2 EDTA-free proteinase inhibitor tablets (Roche Diagnostics GmbH, Mannheim, Germany) per 100 mL of buffer solution in MQ water] by soft vortexing. This was followed by addition of 1.6 mL of 2.5× sodium dodecyl sulfate (SDS)-sample buffer [SDS-SB, 62 mM Tris (pH 6.8) containing 10% (v/v) glycerol, 2.5% (w/v) SDS, and 5% (v/v) 2-ME, pH 6.8], vortexing, sonication (ca. 10 min), and boiling for 1 min at 95 °C. Following centrifugation at 15 000 rpm for 15 min at 4 °C, the proteins in the supernatant were precipitated using a Protein Precipitation Kit (Calbiochem, Darmstadt, Germany). The pellet was air-dried and resolubilized in lysis buffer [LBTT, 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 18 mM Tris-HCl Journal of Proteome Research • Vol. 7, No. 8, 2008 3195

research articles

Figure 2. The experimental strategy and proteomics workflow. Collected RJ (described in Figure 1) was solubilized in homogenization buffer (HB) and sodium dodecyl sulfate (SDS)-sample buffer (SDS-SB) by vortexing and sonication. After boiling and centrifugation of the homogenates, total protein was precipitated and the pellet was solubilized in lysis buffer containing thiourea and Tris (LB-TT). The clear supernatant was used for estimation of protein amount followed by 1-DGE and 2-DGE analyses. The separated proteins on gel were stained with either Coomassie brilliant blue (CBB R-250) or silver nitrate. The stained protein bands/spots were excised from gels and in-gel tryptic digested for analyses by nESI-LC-MS/MS. Proteins were identified using MASCOT search engine and Swiss-Prot/NCBI nonredundant protein databases, and the identified proteins were listed on the RJP (royal jelly proteome) database.

(pH 8.0), 14 mM Trizma base, two EDTA-free proteinase inhibitor cocktail tablets in a final volume of 100 mL buffer, 0.2% (v/v) Triton X-100 (R), containing 50 mM dithiothreitol (DTT)12]. After centrifugation at 15 000 rpm for 15 min at 4 °C, the supernatant was used for protein quantification using the Coomassie Plus protein assay kit (PIERCE Biotechnology, Rockford, IL), and the supernatant was stored in aliquots at -80 °C until further analysis by 1- and 2-DGE. 2.3. 1-DGE. One milliliter of the total soluble protein solution obtained in the previous step (section 2.2) was mixed with 0.4 mL of SDS-SB and a drop of bromophenol blue (BPB). After incubation on the bench (ambient room temperature of 25 °C) for 10 min, the mixture was centrifuged, and the supernatant was used for SDS-polyacrylamide gel electrophoresis (SDSPAGE, 4% T, 2.6% C stacking gels, pH 6.8 and 12.5% T, 2.6% C separating gels, pH 8.8). For detection with silver nitrate (Plus One Silver Staining Kit Protein; GE Healthcare) a total of 5 µg of protein was loaded per sample/well. For detection using 3196

Journal of Proteome Research • Vol. 7, No. 8, 2008

Furusawa et al. Coomassie brilliant blue (CBB) R-250, 40 µg of protein was loaded per sample/well. SDS-PAGE was carried out using PAG on a Nihon Eido (Tokyo, Japan) vertical electrophoresis unit at constant current of 40 mA for ca. 3 h. The running buffer was composed of 0.025 M Tris, 0.192 M glycine, and 0.2% (w/ v) SDS. Molecular mass markers (2.5 µL of the commercially available “ready-to-use” Precision Plus Protein Standards, Dual Color, Bio-Rad, Hercules, CA) were loaded in the well adjacent to the samples. 2.3.1. MS Analysis and Protein Identification. The CBB stained gel with the 1-48 h RJ protein sample was sliced into 14 pieces (see Figure 3), whereas the 2-48 h RJ separated proteins were selectively excised at the two differentially expressed (over the first collection sample) band regions. The individual pieces of the gel matrix were digested with 1 µg of trypsin at 37 °C for 18 h. The tryptic peptide samples were separated by C18 reverse-phase column and analyzed on a nano electrospray ionization mass spectrometer (nESI-LC-MS/ MS). Ultimate nanoLC systems, combined with the FAMOS autosampler and Switchos column switching valve (LC-Packings, Amsterdam, Netherlands), was used. The samples were loaded onto a precolumn (2 cm × 200 µm i.d.; Zorbax 300SBC18, 5 µm, Agilent, CA), and washed with the loading solvent (H2O/0.1% formic acid, flow rate: 4 µL/min.) for 10 min to remove salts. Subsequently, a Switchos II column switching device transferred flow paths to the analytical column (15 cm × 75 µm i.d.; Zorbax 300SB-C18, 5 µm, Agilent). The nanoflow eluted at a flow rate of 200 nL/min using a 110 min gradient elution from 0% solvent A to 32% solvent B, where solvent A was 0.1% formic acid with 5% acetonitrile and solvent B was 0.1% formic acid with 90% acetonitrile in water. The column outlet was coupled directly with the high voltage ESI source, which was interfaced to the QSTAR mass spectrometer (Applied Biosystems, Foster city, CA). The nanospray voltage was typically 2.3 kV in the nESI-LC-MS/MS mode. The nESI-LC-MS/ MS running on the QSTAR instrument was acquired in ‘Information Dependent Acquisition’ mode, which allows the user to acquire MS/MS spectra based on an inclusion mass list and dynamic assessment of relative ion intensity. The data acquisition time was set to 3 s per spectrum over mass/charge (m/z) range of 400-1500 Da. AnalystQS software (Version 1.1; Applied Biosystems, Foster City, CA) was used for generating peaklist. Acquired data were searched against the National Center for Biotechnology Information (NCBI) nonredundant whole protein database (nrdb90) using the MASCOT software package (Version 2.1, Matrix Sciences, U.K.; www.matrixscience.com). The NCBInr database contained 2 821 560 sequences and 968 325 167 residues, and the taxonomy was all entries. Parameters used were peptide mass tolerance (1.0 Da), MS/MS ion mass tolerance (0.8 Da), allowance of two tryptic miscleavages, differential modification (methionine as oxidation), and default selection of charge states ions (only +2, +3). The cutoff score/expectation value for accepting individual MS/MS spectra was 54, which indicates identity or extensive homology of probability lower than 0.05 (p < 0.05). The threshold employed was -10 Log(P), where P is the probability that the observed match is a random event; protein scores are derived from ions scores as a nonprobabilistic basis for ranking protein hits. If peptides matched to multiple members of a protein family, we focused on the unique peptide of identified proteins. The unique peptide of each protein was manually selected to reduce the multimatching peptide mistake, and then MSQuant 1.4 (www.cebi.sud.dk)

RJ Proteomics Reveals Novel RJ Proteins and Phospho/Glycoproteins program was used to regenerate the protein identification list. The MSQuant 1.4 program eliminates peptides having scores lower than applied cutoff score of 54, thereby helping in unambiguous identification of isoforms/individual members of a protein family. Multiple proteins IDs having the same accession number were considered as a single nonredundant protein for the protein list provided in this study. The peptide sequences were listed along with the corresponding proteins (Tables 1 and 2). 2.4. 2-DGE and Protein Visualization. 2-DGE was carried out using precast IPG strip gel on an IPGphor unit (GE Healthcare) followed by the second dimension using ExcelGel XL SDS PAG (gradient 12-14%) on a Multiphor II horizontal electrophoresis unit (GE Healthcare). The volume carrying 100 µg of total soluble protein was mixed with LB-TT containing 0.5% (v/v) pH 4-7 IPG buffer to bring to a final volume of 450 µL. A trace of BPB was added and the sample was centrifuged at 15 000 rpm for 15 min followed by pipetting into 24 cm strip holder tray placed into the IPGphor unit. The IPG strip (pH 4-7; 24 cm, GE Healthcare) was carefully placed over the protein sample avoiding air bubbles between the sample and the gel. The IPG strip was allowed to passively rehydrate with the protein sample for 1.5 h, followed by overlaying the IPG strip with cover fluid (mineral oil), and this was directly linked to a five-step active rehydration and focusing protocol as described previously.13 The whole procedure was controlled at 20 °C, and a total of 76 908 Vh was used for the 24 cm strip. The strip gel was incubated in equilibration buffer [EB, 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS] containing 2% (w/v) DTT for 10 min (twice) with gentle agitation, followed by incubation in the same EB supplemented with 2.5% (w/v) iodoacetamide for the same time periods as above at room temperature. The IPG strip was then placed onto the SDS-PAG. The protein standards (2.5 µL) were loaded on filter papers (GE Healthcare) placed on the PAG adjacent to the pH 4.0 end of the IPG strip. Electrophoresis was carried out at 40 mA as per the instructions described in the manual accompanying the ExcelGel. Three gel replications were performed for each sample. To visualize protein spots, the PAG was stained with silver nitrate (Plus One Silver Staining Kit). Protein patterns in the gels were recorded as digitalized images (24 bit color, resolution 300 dpi) using the ProteomeScan2000 digital scanner (System Biotics KK) and saved as TIFF files. The gels were quantitated in profile mode as instructed in the operating manual of the ImageMaster 2D Platinum software ver. 5.0 (GE Healthcare). To analyze artifact spots, relative ratio of spot volume over the background of gels were calculated using the software. These results were also manually confirmed. 2.4.1. Spot Excision and In-Gel Digestion. The silver nitrate stained protein spots were excised from the 2-D gels using a gel picker (One Touch Spot Picker, P2D1.5, The Gel Company, San Francisco, CA), and transferred to sterile 1.5 mL microcentrifuge tubes. The protein spots were washed twice with 100 mM ammonium bicarbonate (pH 8.5; hereafter called AMBIC) and then dehydrated with acetonitrile. The gel pieces were reduced with 10 mM DTT at 56 °C for 45 min and alkylated with 50 mM iodoacetamide for 45 min at room temperature in the dark in AMBIC solution. Gel pieces were washed with 20 mM AMBIC, dehydrated with acetonitrile, and air-dried. Gel pieces were subjected to in-gel trypsin digestion with 20 µL of 20 mM AMBIC containing 10 ng/µL sequence grade modified trypsin (17 000 U/mg; Promega, Madison, WI) at 4 °C for 10

research articles

min. After removing the supernatant, 10 µL of 20 mM AMBIC was added and incubated at 37 °C for 18 h. The digested solution was transferred into a new microtube. The peptides were extracted from the gel pieces with 30 µL of 0.5% trifluoroacetic acid (TFA) in 50% acetonitrile twice. The extracted peptides were combined in the same microtube and concentrated to 20 µL using a centrifugal concentrator (CC-105; TOMY, Tokyo, Japan). The resulting tryptic peptides were analyzed on the LCQ Deca linear ion trap MS (Thermo Electron). 2.4.2. MS Analysis and Protein Identification. The peptides in 20 µL were used for mass spectral analysis by LC-MS/MS through a nESI source. Briefly, online capillary LC included a monolithic reverse-phase trap column (0.2 mm × 5 cm, MonoCap for fast-flow, GL Science, Tokyo, Japan) and a fastequilibrating C18 capillary column (monolith-type column; i.d, 0.1 mm; length, 50 mm; GL Science). Sample was loaded onto peptide traps for concentration and desalting prior to final separation by C18 column using a linear acetonitrile gradient ranging from 5% to 65% solvent B [H2O/acetonitrile/formic acid, 10/90/0.1 (v/v)] in solvent A [H2O/acetonitrile/formic acid, 98/2/0.1 (v/v)] for a duration of 40 min. The m/z ratios of eluted peptides and fragmented ions from fused-silica Fortis Tip emitter (150 µm o.d., 20 µm i.d.; AMR, Inc., Tokyo, Japan) were analyzed in the data-dependent positive acquisition mode on LC-MS/MS. Following each full scan (400-2000 m/z), a datadependent triggered MS/MS scan for the most intense parent ion was acquired. The heated fused-silica Fortis Tip emitter was held at ion sprays of 1.8 kV and a flow rate of 300 nL/min. Xcalibur Version 1.1 (Thermo Fisher Scientific K.K.) software was used for generating peaklist. Acquired data were searched against the NCBInr whole protein database (nrdb90) using the MASCOT software package (Version 2.1, Matrix Sciences). The NCBInr database (20060909) contained 3 947 950 sequences and 1 358 419 857 residues, and the taxonomy was all entries. Parameters used were peptide mass tolerance (0.8 Da), MS/ MS ion mass tolerance (0.8 Da), allowance of one tryptic miscleavage, differential modification (methionine as oxidation) and static modification (cysteine as carbamidomethylation), default selection of charge states ions (only +2, +3), and a minimum of two nonredundant peptides. The cutoff score/ expectation value for accepting individual MS/MS spectra was 39, which indicates identity or extensive homology of probability lower than 0.05 (p < 0.05). The threshold employed was -10 Log(P), where P is the probability that the observed match is a random event; protein scores are derived from ions scores as a nonprobabilistic basis for ranking protein hits. The peptide sequences were listed along with the corresponding proteins (Table 3). 2.5. Detection of Potential PTMs. For detecting potential PTMs, the 1-D and 2-D gels were stained with fluorescent stains that detect phosphoproteins (ProQ Diamond phosphoprotein gel stain, ProQ DPGS14), glycoproteins (ProQ Emerald glycoprotein gel stain, ProQ EGGS), and total protein (SYPRO Ruby) obtained from Molecular Probes, Inc. (Eugene, OR). The stained gels were visualized on a 245/312 nm UV transilluminator. The details of the staining protocols are mentioned in Supplementary Table 1. For identification of the stained phosphoproteins, the spots were cut out under UV light and processed for in-gel digestion and protein identification as described in sections 2.4.1 and 2.4.2. Journal of Proteome Research • Vol. 7, No. 8, 2008 3197

3198

score

590

398

337

116

114

96

74

56

286

281

137

peptide

21

22

9

3


3

2

3

1

8

7

3

gi|31979

gi|58585098

gi|763431

gi|58585138

gi|62198227

gi|1346640

gi|6448461

gi|189036

gi|110776423

gi|58585108

gi|58585098

acc. no

Histone H2A.2

Major Royal Jelly Protein 1

similar to Human Albumin

Major Royal Jelly Protein 7 Major Royal Jelly Protein 5

PREDICTED: similar to Yellow-h CG1629-PA, partial Nonmuscle Myosin Heavy Chain (NMHC) Glucose Oxidase Myosin-10



protein name

10.21

5.1

5.69

5.95

4.9

5.44

6.48

5.23

5.12

6.83

5.1

pI

13907.08

48886.09

52081.65

70235.97

50540.92

228939.1

67938.03

145084.6

22539.62

51073.52

48886.09

MW

H. sapiens

A. mellifera

Fraction 2 H. sapiens

A. mellifera

A. mellifera

A. mellifera H. sapiens

Homo sapiens

A. mellifera

A. mellifera

Fraction 1 A. mellifera

species

DNA Binding [Compacting DNA Strands]

Unknown [Nutrition]

Binding

Unknown [Nutrition]

Unknown [Nutrition]

Carbohydrate Metabolism Cytoskeleton

Cytoskeleton

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

RHPDYSVVLLLR, VPQVSTPTLVEVSR, DVFLGMFLYEYAR, KVPQVSTPTLVEVSR, AEFAEVSKLVTDLTK, RHPYFYAPELLFFAK, NYAEAKDVFLGMFLYEYAR EYILVLSNK, IMNANVNELILNTR, MVNNDFNFDDVNFR, KVGDGGPLLQPYPDWSFAK, SGVLFFGLVGDSALGCWNEHR, SGVLFFGLVGDSALGCWNEHRTLER, TSDYQQNDIHYEGVQNILDTQSSAK AMGIMNSFVNDIFER, AMGIMNSFVNDIFERIAGEASR

ILGANVNDLIMNTR

ATGVNVLINGR, MGPSYDPMAVVSPR, SGGLMNVERFPYQPPFAWK LQQELDDLTVDLDHQR, DAASLESQLQDTQELLQEETR ILNNDLNFNDINFR, SEYGENKVQYNGVQDVFNTQTTAK

EYILVLSNK, EALPHVPIFDR, IMNANVNELILNTR, YINREYILVLSNK, LTSNTFDYDPKFTK, TVAQSDETLQMIASMK, MVNNDFNFDDVNFR, EALPHVPIFDRYINR, VGDGGPLLQPYPDWSFAK,KVGDGGPLLQPYPDWSFAK, SGVLFFGLVGDSALGCWNEHR, SGVLFFGLVGDSALGCWNEHRTLER, TSDYQQNDIHYEGVQNILDTQSSAK, KVGDGGPLLQPYPDWSFAKYDDCSGIVSASK MTIDGESFTLK, QNLEMVAQNDR, YFDYDFGSEERR, IVNDDFNFDDVNFR, LLKPYPDWSFAEFK, QNLEMVAQNDRTLQMIAGMK, DKTFVTILRYDGVPSTLNVISGK, SQFGENNVQYQGSEDILNTQSLAK, SLNVIHEWKYFDYDFGSEERR, NGVLFVGLVGNSAVGCWNEHQSLQR, QAAIQSGEYDHTKNYPFDVDQWR LTVAGESFTVK, IMENLPQSGR, IINNDFNFNDVNFR, INDPEGNEYMLALSNR, VGDGGPLLQPYPDWSFAK, KVGDGGPLLQPYPDWSFAK, KVGDGGPLLQPYPDWSFAKYDDCSGIVSASK, ESFDVVAQNEETLQMIVSMKIMENLPQSGR LQQELDDLLVDLDHQR, DFSALESQLQDTQELLQEENR, DFSALESQLQDTQELLQEENRQK

matched amino acid sequence

Table 1. A List of Royal Jelly Proteins (1st collection at 48 h; wild cherry and dandelion, etc., were blooming) Separated by 1-DGE and Identified by nESI-LC-MS/MSa

research articles Furusawa et al.

63

63

2

1

57

66

4

2

72

2

58

79

3

2

107

2

62

120

4

2

129

score

3

peptide

Table 1. Continued

gi|2627260

gi|47228503

gi|19527172

gi|149617802

gi|8925552

gi|29888

gi|6981236

gi|21703262

gi|32450747

gi|58585108

gi|110776423

acc. no

Unnamed Protein product

Unnamed Protein product

PREDICTED: similar to Histone H2B isoform 2 Smooth Muscle CellAssociated Protein-1

Light-Harvesting complex Protein LHCG4

PREDICTED: similar to Yellow-h CG1629-PA, partial Major Royal Jelly Protein 2 Anxal-Prov Protein (Annexin A1) Cytoplasmic Actin 2 Myosin, Heavy Polypeptide 9 unnamed protein product

protein name

10.41

6.56

6.01

10.56

5.47

9.19

5.49

4.85

84310.19

248941.6

103450.2

27606.33

36090.21

10937.82

226338.1

17502.09

37635.86

51073.52

6.83

6.2

22539.62

MW

5.12

pI

Cytoskeleton

Cytoskeleton

Calcium and Phospholipid Binding

Unknown [Nutrition]

Unknown [Nutrition]

functional category

Dictyostelium discoideum

Tetraodon nigroviridis

Mus musculus

Binding [cell differentiation; development; protein folding; striated muscle development] Unknown [region:Phosphoinositide 4-kinase accessory/ catalytic domain; phosphatidylinositol cycle, Phosphoinositide 3-kinase catalytic domain; cell motility, the Ras pathway, vesicle trafficking and secretion, and apoptosis] Unknown

Unknown [region: S-100/ICaBP-like domain; S-100/ intestinal calcium binding domain] Bigelowiella Photosynthesis natans [light-harvesting [Chlorarachnion complex protein, CCMP621] Chlorophyll A-B binding protein] Ornithorhynchus DNA Binding anatinus [Compacting DNA Strands]

H. sapiens

Boltenia villosa Rattus norvegicus

Xenopus laevis

A. mellifera

A. mellifera

species


TVVIAVNHK, KELIIKVLLNILR

HVAAIGPRFR, KSQPSMYEQLR

KLLAAGVVSAMTCMVK, RAAAGGLAMLTSMRPTLCSR

MNSFVNDIFERIAGEASR

TGAITNKLVK, VNGGPAGEGLDAINPGGAFDPLGLADDPDTFAELKVK

ALNSIIDVYHK, MLTELEKALNSIIDVYHKYSLIK

LDLAGRDLTDYLMKILTER, TTGIVMDTGDGVSHTVPIYEGYALPHAIIR QLLQANPILEAFGNAK, DLGEELEALKTELEDTLDSTAAQQELR

GVDEATIIDILTKR, GLGTDEDTLIEILASR

QNLEMVAQNDRTLQMIAGMK, SQFGENNVQYQGSEDILNTQSLAK, SLNVIHEWKYFDYDFGSEERR

IINNDFNFNDVNFR, INDPEGNEYMLALSNR, KVGDGGPLLQPYPDWSFAK

RJ Proteomics Reveals Novel RJ Proteins and Phospho/Glycoproteins

research articles

Journal of Proteome Research • Vol. 7, No. 8, 2008 3199

3200

56

55

743

531

406

235

2

31

25

19

7

score

1

peptide

Table 1. Continued


gi|58585098

gi|42601246

gi|6448461

gi|58585138

gi|145580626

gi|5738567

acc. no

Major Royal Jelly Protein MRJP5 precursor Major Royal Jelly Protein 1

Glucose Oxidase


E2F Transcription Factor 7

POL Protein

protein name

5.1

8.66

6.48

5.95

8.33

8.48

pI

48886.09

68289.46

67938.03

70235.97

99888.13

113470.5

MW

functional category

A. mellifera

Apis cerana

A. mellifera


Unknown [Nutrition]

Unknown [Nutrition]

Carbohydrate Metabolism

Unknown [Nutrition]

Human Synthesis??? immunodeficiency [aspartic-type virus 1 endopeptidase activity, DNA binding, integrase activity, ribonuclease H activity, RNA binding, RNA-directed DNA polymerase activity, zinc ion binding, DNA integration, proteolysis, RNA-dependent DNA replication] H. sapiens Transcription [regulation of progression through cell cycle; regulation of transcription, DNA-dependent]

species

EYILVLSNK, IMNANVNELILNTR, MVNNDFNFDDVNFR, TVAQSDETLQMIASMK, TSDYQQNDIHYEGVQNILDTQSSAK, LAIDKCDRLWVLDSGLVNNTQPMCSPK

GMTFVTVPR, LANSMNVIHEWK, QMNEYMMALSMK, YKGVPSSLNVISEK, YLDYDFGSDERR, ILGANVNDLIMNTR, LTSNTFDYDPKYIK, GVPSSLNVISEKIGNGGR, FINNDYNFNEVNFR, QMNEYMMALSMKLQK, MNRQMNEYMMALSMK, LQKFINNDYNFNEVNFR, MNRQMNEYMMALSMKLQK, ENMDMVAQNEETLQTVVAMK, SEYGANNVQYQGVQDIFNTESIAK, KLANSMNVIHEWKYLDYDFGSDER, LANSMNVIHEWKYLDYDFGSDERR, SEYGANNVQYQGVQDIFNTESIAKIMSK, IAIDKFDRLWILDSGIINNTQPMCSPK ATGVNVLINGR, MSALNVQPTSK, DLGVEFQKIELK, SGGLMNVERFPYQPPFAWK, EVILSAGSVNTPQLLMLSGIGPK, REVILSAGSVNTPQLLMLSGIGPK, VADASVQPQVISGNPVASVNMVGER, ITLNSKDPLDPPVIWSNDLATEHDR, EVILSAGSVNTPQLLMLSGIGPKEHLR, GLRVADASVQPQVISGNPVASVNMVGER, AAEEAGFGVSEDLSGDRINGFTVAQTISR, REVILSAGSVNTPQLLMLSGIGPKEHLR, GRITLNSKDPLDPPVIWSNDLATEHDR, ITLNSKDPLDPPVIWSNDLATEHDRSVMIQAIR QMNEYMMALSMK, YKGVPSSLNVISKK, LHVFDLNTSQQIK, LTSNTFDYDPKYIK, FINNDYNFNEVNFR, QMNEYMMALSMKLQK, MNRQMNEYMMALSMKLQK, QNMDMVAQNEETLQTLQTVVAMK

RLEIPSGGAD, QKSLGLLCQKFLAR

RGDSPLPEAGTEGK



62

55

55

3

1

1

54

174

6

2

177

5

54

213

18

3

score

peptide

Table 1. Continued

gi|118085525

gi|4574723

gi|23126818

gi|114669123

gi|4433771

gi|110776423

gi|110756961

gi|66547819

acc. no

Putative Replication Protein E1 PREDICTED: similar to Voltage-Gated Sodium Channel alpha subunit

PREDICTED: similar to Major Royal Jelly Protein MRJP5 PREDICTED: similar to Glucose Dehydrogenase isoform 1 PREDICTED: similar to Yellow-h CG1629-PA, partial MHC class II beta 1 PREDICTED: myosin, heavy polypeptide 10, nonmuscle isoform 1 COG1020: Non-Ribosomal Peptide Synthetase Modules and Related Proteins

protein name

6.13

5.35

4.78

5.47

5.56

5.12

5.58

5.71

pI

181073.3

73310.46

122022.1

227996.5

8005.06

22539.62

69349.46

33707.13

MW

Cytoskeleton

Defense

Unknown [Nutrition]

Unknown [Carbohydrate Metabolism]

Unknown [Nutrition]

functional category

Unknown [This domain catalyzes a condensation reaction to form peptide bonds in nonribosomal peptide biosynthesis, Secondary metabolites biosynthesis transport, and catabolism, AMP-binding enzyme, acyl-CoA synthase, Phosphopantetheine attachment site, attachment of activated fatty acid and amino-acid] Human Unknown [region:E1 papillomavirus Protein, candHPV85 Papillomavirus helicase] Gallus Unknown [region: Ion gallus transport] (red jungle fowl)

Nostoc punctiforme PCC 73102

Pan troglodytes

Salvelinus namaycush

A. mellifera

A. mellifera

A. mellifera

species


NLDESQTSSSASLPPSYDSVTR, SLGIEECEEEQFPKSQLTDSQRK

LLHALKR, KYGAHSTENSPCR

QLLAGGDVLSVTHVQK

DAASLESQLQDTQELLQEETR

HNAAIYYSAILDK, NAETWNKGSELAQELGQLESYCKHNAAIYYSAILDK

IINNDFNFNDVNFR, INDPEGNEYMLALSNR, NGVLFLGLVGNSGIACVNEHQVLQR, IINNDFNFNDVNFRILGANVDDLMR, LAIDKCDRLWVLDSGLVNNTQPMCSPK

IALSLSNTSALAR, KYHSTGGLLNVER, VTSGNTAAPAIMIGER, LASNDPFAKPVIHGNYLSDPMDEAVLLHGIR

LTSNTFDYDPKYIK, IAIDKFDRLWILDSGIINNTQPMCSPK, MMAAGESFTAQDGIFGMALSPMTNNLYYSPLSSR, YIKMMAAGESFTAQDGIFGMALSPMTNNLYYSPLSSR, LTSNTFDYDPKYIKMMAAGESFTAQDGIFGMALSP MTNNLYYSPLSSR


research articles


3202

54

902

230

225

169

69

63

58

48

5

6

6

5

2

2

score

2

peptide

Table 1. Continued


gi|23271256

gi|15620867

gi|66547819

gi|58585138

gi|58585098

gi|6448461

gi|56422035

gi|33562986

acc. no

Major Royal Jelly Protein 1 Major Royal Jelly Protein 5 PREDICTED: similar to Major Royal Jelly Protein MRJP5 KIAA1904 Protein Gametogenetin

Glucose Oxidase


ORF18

protein name

10.3

7.63

5.71

5.95

5.1

6.48

6.87

5.64

pI

66698.52

89759.99

33707.13

70235.97

48886.09

67938.03

65695.81

58510.16

MW

H. sapiens

H. sapiens

A. mellifera

A. mellifera

A. mellifera

A. mellifera


Comamonas testosteroni

species

Unknown

Unknown

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]


Unknown [Nutrition]

Unknown [region: necessary for testosterone degradation in TA441, Acyl-CoA synthetases (AMP-forming)/ AMP-acid ligases II. Lipid metabolism/ Secondary metabolites biosynthesis, transport, and catabolism, Lipid metabolism]

functional category matched amino acid sequence

FNGAGGGIGAPR, LTRGLGVWFPGSATPPGLMVPR

EEVYMAAGHALRK, EEVYMAAGHALRKK

LTSNTFDYDPKYIK, MMAAGESFTAQDGIFGMALSPMTNNLYYSPLSSR,

ILGANVNDLIMNTR, LTSNTFDYDPKYIK, FINNDYNFNEVNFR, SEYGANNVQYQGVQDIFNTESIAK

DKTFVTIER, LTVAGESFTVK, LLTFDLKTSK, LTSNTFDYDPR, HIDFDFGSDERR, LLTFDLKTSKLVK, YTKLTVAGESFTVK, GGPLLRPYPDWSFAK, IINNDFNFNDVNFR, INDPEGNEYMLALSNR, GKGGPLLRPYPDWSFAK, MQKIINNDFNFNDVNFR, SGEFDHTKNYPFDVDRWR, ESFDVVAQNEETLQMIVSMK, VIYEWKHIDFDFGSDERR, NGVLFLGLVGNSGIACVNEHQVLQR, LTSNTFDYDPRYTKLTVAGESFTVK, IINNDFNFNDVNFRILGANVDDLMR, IMENLPQSGRINDPEGNEYMLALSNR, GGPLLRPYPDWSFAKYEDCSGIVSAFK, VVSKNGVLFLGLVGNSGIACVNEHQVLQR, IAVDKFDRLWVLDSGLVNNNQPMCSPK, TNTMVYIADEKGEGLIMYQNSDDSFHR, GKGGPLLRPYPDWSFAKYEDCSGIVSAFK AADFIKEDWGELLQLL, SGGLMNVERFPYQPPFAWK, REVILSAGSVNTPQLLMLSGIGPK, ITLNSKDPLDPPVIWSNDLATEHDR, REVILSAGSVNTPQLLMLSGIGPKEHLR EYILVLSNK, IMNANVNELILNTR, MVNNDFNFDDVNFR, TVAQSDETLQMIASMK, SGVLFFGLVGDSALGCWNEHRTLER, TSDYQQNDIHYEGVQNILDTQSSAK

IKPDDTADLMFTSGTTGRPK, AAIVENGKSISYAQLQQLSR


137

106

96

61

4

3

2

428

16

4

584

35

358

886

43

11

score

peptide

Table 1. Continued

gi|89045073

gi|58585108

gi|66511551

gi|6448461

gi|58585170

gi|58585098

gi|110776423

gi|56422035

acc. no

PREDICTED: similar to Kinase suppressor of ras-1 (Kinase suppressor of ras)


PREDICTED: similar to Glucocerebrosidase precursor isoform 2

Glucose Oxidase



PREDICTED: similar to Yellow-h CG1629-PA, partial


protein name

8.58

6.83

5.19

6.48

5.89

5.1

5.12

6.87

pI

24141.85

51073.52

57952.35

67938.03

52915.46

48886.09

22539.62

65695.81

MW

H. sapiens

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera


species

Unknown

Unknown [Nutrition]

Unknown [region: O-Glycosyl hydrolase


Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

DKTFVTIER, LTVAGESFTVK, ILGANVDDLMR, HIDFDFGSDERR, YTKLTVAGESFTVK, GGPLLRPYPDWSFAK, IINNDFNFNDVNFR, NNGVPSSLNVVTNKKGK, INDPEGNEYMLALSNR, GKGGPLLRPYPDWSFAK, MQKIINNDFNFNDVNFR, ESFDVVAQNEETLQMIVSMK, VIYEWKHIDFDFGSDERR, NGVLFLGLVGNSGIACVNEHQVLQR, LTSNTFDYDPRYTKLTVAGESFTVK, QVEIPHNIAVNATTGMGELVSLAVQAIDR, GGPLLRPYPDWSFAKYEDCSGIVSAFK, VVSKNGVLFLGLVGNSGIACVNEHQVLQR, IAVDKFDRLWVLDSGLVNNNQPMCSPK, TNTMVYIADEKGEGLIMYQNSDDSFHR LTVAGESFTVK, ILGANVDDLMR, YTKLTVAGESFTVK, IINNDFNFNDVNFR, INDPEGNEYMLALSNR, VGDGGPLLQPYPDWSFAK, KVGDGGPLLQPYPDWSFAK, MQKIINNDFNFNDVNFR, ESFDVVAQNEETLQMIVSMK, SLPILHEWKFFDYDFGSDER, NGVLFLGLVGNSGIACVNEHQVLQR, LTSNTFDYDPRYTKLTVAGESFTVK, VVSKNGVLFLGLVGNSGIACVNEHQVLQR, KVGDGGPLLQPYPDWSFAKYDDCSGIVSASK EYILVLSNK, IMNANVNELILNTR, LTSNTFDYDPKFTK, MVNNDFNFDDVNFR, TVAQSDETLQMIASMK, VGDGGPLLQPYPDWSFAK, KVGDGGPLLQPYPDWSFAK, SGVLFFGLVGDSALGCWNEHR, SLPILHEWKFFDYDFGSDER, SGVLFFGLVGDSALGCWNEHRTLER, TSDYQQNDIHYEGVQNILDTQSSAK, TSDYQQNDIHYEGVQNILDTQSSAKVVSK, KVGDGGPLLQPYPDWSFAKYDDCSGIVSASK QAAIQSGEYDR, QVEIPHDVATTGK, NEDTLQMVVSMK, SENQGNDVQYER, QAAIQSGEYDRTK, WKYLDYDFDNDERR, QNIDVVARNEDTLQMVVSMK, SENQGNDVQYERVQDVFDSQLTVK, LLQPYPDWSFAKYEDCSGIVSAHK, NEYLLALSDRNQNVLNNDLNLEHVNFQILGANVNDLIR MGPSYDPMAVVSPR, AADFIKEDWGELLQLL, SGGLMNVERFPYQPPFAWK, AAEEAGFGVSEDLSGDRINGFTVAQTISR NGVDIWGVSTGNEPFDAYIPFER, KAVELNPDLRFFSAAWSAPTWMK, AYTLDDYDDDATLQHFALAPEDVEYKIPYARK, DGGPTYINNNVDSPIIVNPENDEFYKQPMYYALK IVNDDFNFDDVNFR, QNLEMVAQNDRTLQMIAGMK, SLNVIHEWKYFDYDFGSEERR KEGGGGGDAGAAEGGSGVAPSR, KEGGGGGDAGAAEGGSGVAPSRVLQQCGQLQK



research articles


3204

1000

297

90

750

335

10

2

40

12

59

score

53

2

peptide

Table 1. Continued


gi|58585098

gi|58585108

gi|58585108

gi|62198227

gi|58585098

gi|71738784

acc. no






ACP54A1

protein name

5.1

6.83

6.83

4.9

5.1

9.58

pI

48886.09

51073.52

51073.52

50540.92

48886.09

5149.55

MW

A. mellifera


A. mellifera

A. mellifera


Drosophila melanogaster

species

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown

functional category matched amino acid sequence

TLQMIAGMK, SLNVIHEWK, ILGANVKELIR, QNLEMVAQNDR, IVNDDFNFDDVNFR, MQKIVNDDFNFDDVNFR, IAIDKFDRLWVLDSGLVNR, TFVTILRYDGVPSTLNVISGK, QNLEMVAQNDRTLQMIAGMK, TSNHLKQIEIPHDIAVNATTGK, IVNDDFNFDDVNFRILGANVK, DKTFVTILRYDGVPSTLNVISGK, SLNVIHEWKYFDYDFGSEER, SQFGENNVQYQGSEDILNTQSLAK, SLNVIHEWKYFDYDFGSEERR, NGVLFVGLVGNSAVGCWNEHQSLQR, MQKIVNDDFNFDDVNFRILGANVK, IKEELPHFVGSNKPVKDEYMLVLSNR, AVSKNGVLFVGLVGNSAVGCWNEHQSLQR, GGLVSLAVQAIDLANTLVYMADHKGDALIVYQ NADDSFHR LTSNTFDYDPKFTK, MVNNDFNFDDVNFR, TVAQSDETLQMIASMK, EALPHVPIFDRYINR, VGDGGPLLQPYPDWSFAK, SGVLFFGLVGDSALGCWNEHR, SGVLFFGLVGDSALGCWNEHRTLER, TSDYQQNDIHYEGVQNILDTQSSAK, TSDYQQNDIHYEGVQNILDTQSSAKVVSK

EYILVLSNK, EALPHVPIFDR, EYILVLSNKMQK, LLTFDLTTSQLLK, IMNANVNELILNTR, LTSNTFDYDPKFTK, MVNNDFNFDDVNFR, TVAQSDETLQMIASMK, EALPHVPIFDRYINR, GEGLIVYHNSDDSFHR, VGDGGPLLQPYPDWSFAK, TVAQSDETLQMIASMKIK, KVGDGGPLLQPYPDWSFAK, KVGDGGPLLQPYPDWSFAK, MQKMVNNDFNFDDVNFR, HNIRTVAQSDETLQMIASMK, SGVLFFGLVGDSALGCWNEHR, VVSKSGVLFFGLVGDSALGCWNEHR, SGVLFFGLVGDSALGCWNEHRTLER, TSDYQQNDIHYEGVQNILDTQSSAK, EALPHVPIFDRYINREYILVLSNK, EYILVLSNKMQKMVNNDFNFDDVNFR, VGDGGPLLQPYPDWSFAKYDDCSGIVSASK, TSDYQQNDIHYEGVQNILDTQSSAKVVSK, KVGDGGPLLQPYPDWSFAKYDDCSGIVSASK, LSSLAVQSLDCNTNSDTMVYIADEKGEGLIVY HNSDDSFHR ILIGGVSDLLENTR, LLQPYPDWSWTK, ILNNDLNFNDINFR, MQKILNNDLNFNDINFR, ENTDMVAQNEETLQMIVGMK, SEYGENKVQYNGVQDVFNTQTTAK, QLLPHIVIIDIDNIINDEYMLVLTNR, ILNNDLNFNDINFRILIGGVSDLLENTR IVNDDFNFDDVNFR, QNLEMVAQNDRTLQMIAGMK

HSCSKLLSLMVLLCLAFDLCPVSAMK


82

64

2

1

55

1

108

77

3

3

151

4

285

212

7

9

321

14

307

372

12

10

167

score

5

peptide

Table 1. Continued

gi|17509865

gi|6448461

gi|58585138

gi|58585098

gi|58585108

gi|71546273

gi|60115688

gi|62198227

gi|110776423

gi|58585108

gi|58585098

gi|62198227

acc. no

Y37E3.8b

Major Royal Jelly Protein 5 Glucose Oxidase



Venom Protein 2 precursor ABC Transporter






protein name

9.63

6.48

5.95

5.1

6.83

5.64

4.51

4.9

5.12

6.83

5.1

4.9

pI

9806.58

67938.03

70235.97

48886.09

51073.52

34471.73

24788.52

50540.92

22539.62

51073.52

48886.09

50540.92

MW

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

A. mellifera A. mellifera Caenorhabditis elegans

A. mellifera


Carbohydrate Metabolism Unknown [region: 50S ribosomal protein L15P]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

A. Unknown mellifera Syntrophobacter Transport [ABC-type fumaroxidans multidrug transport MPOB system, ATPase component Defense mechanisms, Transport, ATP-binding protein]

A. mellifera

A. mellifera

A. mellifera


A. mellifera

species

VLGKGLLPETPLIVK

MTIDGESFTLK, LHVFDLKTSNHLK, YAKMTIDGESFTLK, IVNDDFNFDDVNFR, QNLEMVAQNDRTLQMIAGMK, SLNVIHEWKYFDYDFGSEER, SQFGENNVQYQGSEDILNTQSLAK, SLNVIHEWKYFDYDFGSEERR, NGVLFVGLVGNSAVGCWNEHQSLQR LTSNTFDYDPKFTK, MVNNDFNFDDVNFR, TVAQSDETLQMIASMK, SGVLFFGLVGDSALGCWNEHRTLER, TSDYQQNDIHYEGVQNILDTQSSAK, TSDYQQNDIHYEGVQNILDTQSSAKVVSK, MVNNDFNFDDVNFRIMNANVNELILNTR LHVFDLNTSHQLK, ILGANVNDLIMNTR, FINNDYNFNEVNFR MGPSYDPMAVVSPR, AADFIKEDWGELLQLL

MATLEDVFLR

IMNANVNELILNTR, LTSNTFDYDPKFTK, MVNNDFNFDDVNFR, TVAQSDETLQMIASMK, VGDGGPLLQPYPDWSFAK, SGVLFFGLVGDSALGCWNEHRTLER, TSDYQQNDIHYEGVQNILDTQSSAK, TSDYQQNDIHYEGVQNILDTQSSAKVVSK, KVGDGGPLLQPYPDWSFAKYDDCSGIVSASK LHVFDLK, MTIDGESFTLK, IVNDDFNFDDVNFR, QNLEMVAQNDRTLQMIAGMK, IVNDDFNFDDVNFRILGANVK, SQFGENNVQYQGSEDILNTQSLAK, SLNVIHEWKYFDYDFGSEERR, NGVLFVGLVGNSAVGCWNEHQSLQR, IKEELPHFVGSNKPVKDEYMLVLSNR IINNDFNFNDVNFR, INDPEGNEYMLALSNR, VGDGGPLLQPYPDWSFAK, ESFDVVAQNEETLQMIVSMK, NGVLFLGLVGNSGIACVNEHQVLQR, KVGDGGPLLQPYPDWSFAKYDDCSGIVSASK ILNNDLNFNDINFR, IKQLLPHIVIIDIDNIINDEYMLVLTNR, MTVEGESFTVQDGIYGMALSPMTNNLYYSPLASR, SLAVQAISSVNTLVYIADNKGDGLIVYQNSDDSFHR NVDTVLVLPSIER, KNVDTVLVLPSIER

ILIGGVSDLLENTR, QLLPHIVIIDIDNIINDEYMLVLTNR, IKQLLPHIVIIDIDNIINDEYMLVLTNR, SLAVQAISSVNTLVYIADNKGDGLIVYQNSDDSFHR



research articles


3206

153

103

56

332

238

89

62

56

183

127

115

79

232

97

64

56 56

3

1

18

6

2

2

2

9

4

5

2

6

3

2

1 1

score

5

peptide

Table 1. Continued


gi|17509865 gi|8568805

gi|58585138

gi|48094573

gi|58585098

gi|6448461

gi|58585108

gi|58585098

gi|48094573

gi|66557660

gi|58585138

gi|56422035

gi|58585098

gi|58585108

gi|17509865

gi|56422035

gi|58585098

acc. no

PREDICTED: Hypothetical Protein Major Royal Jelly Protein 5 Y37E3.8b putative ABC Transporter, ATP-Binding subunit


Glucose Oxidase

PREDICTED: Hypothetical Protein Major Royal Jelly Protein 1 Major Royal Jelly Protein 2

Major Royal Jelly Protein 3 Major Royal Jelly Protein 5 PREDICTED: Hypothetical Protein



Major Royal Jelly Protein 3 Y37E3.8b


protein name

9.63 6.44

5.95

6.6

5.1

6.48

6.83

5.1

6.6

5.48

5.95

6.87

5.1

6.83

9.63

6.87

5.1

pI

9806.58 27118.84

70235.97

19433.6

48886.09

67938.03

51073.52

48886.09

19433.6

21348.07

70235.97

65695.81

48886.09

51073.52

9806.58

65695.81

48886.09

MW

A. mellifera C. elegans Streptomyces coelicolor A3

A. mellifera


A. mellifera

A. mellifera A. mellifera


A. mellifera A. mellifera A. mellifera

A. mellifera


A. mellifera C. elegans


species

Unknown Transpotr, ATP-Binding

Unknown [Nutrition]

Unknown

Unknown [Nutrition]


Unknown [Nutrition]

Unknown [Nutrition]

Unknown

Unknown

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [region: 50S ribosomal protein L15P]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

VLGKGLLPETPLIVK MAIISTAGLARTFATRR

ILGANVNDLIMNTR, FINNDYNFNEVNFR

EYILVLSNK, IMNANVNELILNTR, MVNNDFNFDDVNF, TVAQSDETLQMIASMK, TSDYQQNDIHYEGVQNILDTQSSAK EQVANALLEGSSDLIGALK, EQVANALLEGSSDLIGALKDAVELAIR

SADNIVKQFGK, SADNIVKQFGKGFAAVAK, EQVANALLEGSSDLIGALK, EQVANALLEGSSDLIGALKDAVELAIR MVNNDFNFDDVNFR, TVAQSDETLQMIASMK, TSDYQQNDIHYEGVQNILDTQSSAK IVNDDFNFDDVNFR, QNLEMVAQNDRTLQMIAGMK, SQFGENNVQYQGSEDILNTQSLAK MGPSYDPMAVVSPR, VADASVQPQVISGNPVASVNMVGER

QIQEQWNIPDQDTIVK, QAVDIAVQATQNLNNQLQTAATQKS

ILGANVNDLIMNTR, FINNDYNFNEVNFR

QNLEMVAQNDR, IVNDDFNFDDVNFR, QNLEMVAQNDRTLQMIAGMK, IVNDDFNFDDVNFRILGANVK, SQFGENNVQYQGSEDILNTQSLAK, NGVLFVGLVGNSAVGCWNEHQSLQR, MQKIVNDDFNFDDVNFRILGANVK, IKEELPHFVGSNKPVKDEYMLVLSNR EYILVLSNKMQK, IMNANVNELILNTR, MVNNDFNFDDVNFR, TVAQSDETLQMIASMK, SGVLFFGLVGDSALGCWNEHR, TSDYQQNDIHYEGVQNILDTQSSAK IINNDFNFNDVNFR, ESFDVVAQNEETLQMIVSMK

LTSNTFDYDPKFTK, MVNNDFNFDDVNFR, TVAQSDETLQMIASMK, TSDYQQNDIHYEGVQNILDTQSSAK, LLTFDLKTSKLVK, IINNDFNFNDVNFR, ESFDVVAQNEETLQMIVSMK VLGKGLLPETPLIVK



research articles

a Identified proteins from RJ of A. mellifera L. were categorized into fractions. The table lists peptide, score, accession no, protein names, theoritical pI, theoritical mass (kDa), species, functional category and matched amino acid sequence. Polymorphism of the MRJP 3 protein is a consequence of polymorphism of a region with a variable number of tandem repeats located at the C-terminal part of the MRJP3 coding region. So we regard these proteins are same MRJP3. The protein includes these proteins, which were identified as major royal jelly protein 3 (gi|58585142) and royal jelly protein RJP57-1 (gi|1113119) (See Supplementary Figure 1).

Lipid Metabolism 229796.8 5.17 64 1

gi|409450

150 4

gi|58585138

Major Royal Jelly Protein 5 Acetyl-CoA Carboxylase

5.95

70235.97

A. mellifera Cyclotella cryptica

Unknown [Nutrition]

EYILVLSNK, IMNANVNELILNTR, MVNNDFNFDDVNFR, TVAQSDETLQMIASMK, TSDYQQNDIHYEGVQNILDTQSSAK GMTFVTVPR, ILGANVNDLIMNTR, FINNDYNFNEVNFR FGSMIVDSLR Unknown [Nutrition] 191

gi|58585098


5.1

48886.09

Fraction 14 A. mellifera 6

score peptide

Table 1. Continued

acc. no

protein name

pI

MW

species

functional category



3. Results and Discussion 3.1. The Experimental Strategy and Identified RJ Proteins by a Combination of 1-/2-DGE Analyses. The experimental strategy for a systematic analysis of the RJ proteome is presented in Figure 2. We collected RJs, which is a worker honey bee (A. mellifera L.) product, in a time-course manner (Figure 1) and depending on the season; the RJ was sampled at two seasons when different flowering species were in bloom. For extraction of the total RJ proteins, we used HB and SDSSB and the extracted proteins in the supernatant were precipitated and resolubilized in LB-TT to give the total soluble protein extract. This novel extraction protocol not only removes nonprotein contaminants from the complex RJ mixture, but also improves solubilization of the extracted proteins. The protein extract was then used for 1- and 2-DGE analyses. The tryptic peptides either from 1-DGE bands or 2-DGE spots were analyzed by tandem mass spectrometry (nESI-LC-MS/MS). Proteins were identified using Swiss-Prot/NCBI nonredundant protein database and the MASCOT search engine. Using a combination of 1-D and 2-D systems, we could identify a large number of proteins that would be underrepresented using either method alone. Although RJ proteomics analysis data have been reported,9–11 this is a first comprehensive study presenting a standardized protein extraction protocol, the largest number of identified proteins, including novel proteins, and creating a 2-D gel reference map. Furthermore, phosphorylated proteins were shown to be present on both 1- and 2-D gels, a novel finding of the study. Moreover, by time course and seasonal sampling of RJ, we could show for the first time variations in the RJ protein profiles. 1- and 2-DGE together identified a total of 210 proteins, representing 52 nonredundant proteins. 1-DGE and 2-DGE separately identified 47 (including from both the 1-48 h and 2-48 h RJ samples) and 14 nonredundant proteins, respectively, where only 9 proteins were common in both data sets. From this result, it is quite clear that different proteins are present and/or identified only by 1-DGE but not by 2-DGE. In all, there are 42 such nonredundant proteins from 1-DGE. Fourteen of the 42 proteins have a pI value of over 7.0, which are basic in nature and, thus, may not be represented on the 2-D gel. Some other proteins were membrane proteins, which are also not well-represented on 2-D gel, suggesting that 1-DGE and 2-DGE are complementary approaches whose applications result in a more comprehensive proteome investigation. In addition, we visualized the potential protein PTMs using ProQ DPGS/EGGS and SYPRO RUBY staining, and we identified the potential phosphoproteins by MS. The proteomics data, including 2-D map has also been posted to the Web site http:// foodfunc.agr.ibaraki.ac.jp/RJP.html, which we hope will serve as a proteomics resource for the scientific community, especially the honey bee and RJ researchers. 3.2. 1-DGE. Total soluble proteins at 24-72 h after first and second collections were separated on 12.5% SDS-PAG as described in section 2.3 (Figure 3). The proteins (5 µg/lane) in panel A were stained with silver nitrate, and the proteins (40 µg/lane) in panel B were stained with CBB R-250. The molecular weight markers are indicated on the left-hand side of gel 1. The proteins (of first and second collections at 48 h; 3 lanes each) stained with CBB R-250 were separated into 14 regions (marked by red boxes and numbered 1-14) and analyzed for their protein components by LC-MS/MS. Two different (from the first collection) bands in the second collection (labeled 2-4 and 2-5, respectively; marked in blue boxes) were also excised Journal of Proteome Research • Vol. 7, No. 8, 2008 3207

3208

score

616

448

258

130

116

85

68

68

54

peptide

90

58

7


4

3

2

2

2

2

gi|9931616

gi|5858516

gi|4260124

gi|5858510

gi|58585138

gi|48118838

gi|58585098

gi|110776423

gi|56422035

acc. no

sof2460 precursor

Major Royal Jelly Protein 2 Major Royal Jelly Protein MRJP5 Alpha-Glucosidase


PREDICTED: similar to Y4C6B.6




protein name

5.89

5.06

8.66

6.83

5.95

7.03

5.1

5.12

6.87

pI

species

44037.7

65565

68289.5

51073.5

70236

58571

48886.1

22539.6

A. mellifera A. mellifera A. mellifera Streptococcus pyogenes

A. mellifera

A. mellifera

A. mellifera

A. mellifera

Fraction 2-4 65695.8 A. mellifera

MW

LTVAGESFTVK, IMENLPQSGR, ILGANVDDLMR, HIDFDFGSDER, NYPFDVDRWR, HIDFDFGSDERR, GGPLLRPYPDWSFAK, IINNDFNFNDVNFR, INDPEGNEYMLALSNR, GEGLIMYQNSDDSF HR, ESFDVVAQNEETLQMIVSMK, NGVLFLGLVGNSGIACVNEHQVLQR, VVSKNGVLFLGLVGNSGIACVNEHQVLQR, IAVDKFDRLWVLDSGLVNNNQPMCSPK, TNTMVYIADEKGEGLIMYQNSDDSFHR LTVAGESFTVK, IMENLPQSGR, ILGANVDDLMR, IINNDFNFNDVNFR, INDPEGNEYMLALSNR, VGDGGPLLQPYPDWSFAK, KVGDGGPLLQPYPDWSFAK, ESFDVVAQNEETLQMIVSMK, NGVLFLGLVGNSGIACVNEHQVLQR, DPQYEENNVQYEGSQDILNTQSFGK, VVSKNGVLFLGLVGNSGIACVNEHQVLQR EYILVLSNK, IMNANVNELILNTR, TVAQSDETLQMIASMK, VGDGGPLLQPYPDWSFAK, KVGDGGPLLQPYPDWSFAK, SGVLFFGLVGDSALGCWNEHR, TSDYQQNDIHYEGVQNILDTQSSAK LCLELSPQSMNTLK, IIGFGGAFTDATGINIAK, LAVELNPEVLLFAAAWTAPLWMK, TGGPTVVNNNLDAPIIVNPETDEFYKQPLYYAIK


Unknown [cation binding, glucosylceramidase activity] Unknown ILGANVNDLIMNTR, [Nutrition] ENMDMVAQNEETLQTVVAMK, SEYGANNVQYQGVQDIFNTESIAK Unknown LHVFDLKTSNHLK, IVNDDFNFDDVNFR [Nutrition] Unknown LHVFDLNTSQQIK, QNMDMVAQNEETLQTVVAMK [Nutrition] Carbohydrate VSALGFFILISQDAK, Metabolism LVDNWMTYMPPSGIPNWVPGNHDQLR Unknown EISDPIDISGKTDDDLDQLLDELR, [region: von LTEVTTSNPLLHWPPIFNHTNRK Willebrand factor (vWF) type A domain]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

Table 2. A list of Royal Jelly Proteins (2nd collection at 48 h; R. pseudoacacia L. were blooming) Separated by 1-DGE and Identified by nESI-LC-MS/Ma


56

1

gi|\ill\

gi|5858510

gi|58585098

gi|58585170

gi|110776423

gi|56422035

acc. no






protein name

5.95

6.83

5.1

5.89

5.12

6.87

pI

species

70236

51073.5

48886.1

52915.5

22539.6

A. mellifera A. mellifera

A. mellifera

A. mellifera

A. mellifera

Fraction 2-5 65695.8 A. mellifera

MW

Unknown [Nutrition] Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

SEYGANNVQYQGVQDIFNTESIAK

LTVAGESFTVK, ILGANVDDLMR, TNTMVYIADEK, LTSNTFDYDPR, HIDFDFGSDERR, GGPLLRPYPDWSFAK, IINNDFNFNDVNFR, INDPEGNEYMLALSNR, HIDFDFGSDERRDAAIK, ESFDVV AQNEETLQMIVSMK, VIYEWKHIDFDFGSDERR, NGVLFLGLVGNSGIACVNEHQVLQR, IMENLPQSGRINDPEGNEYMLALSNR, QVEIPHNIAVNATTGMGELVSLAVQAIDR, GGPLLRPYPDWSFAKYEDCSGIVSAFK, IAVDKFDRLWVLDSGLVNNNQPMCSPK, TNTMVYIADEKGEGLIMYQNSDDSFHR LTVAGESFTVK, ILGANVDDLMR, LTSNTFDYDPR, IINNDFNFNDVNFR, INDPEGNEYMLALSNR, VGDGGPLLQPYPDWSFAK, KVGDGGPLLQPYPDWSFAK, ESFDVVA QNEETLQMIVSMK, SLPILHEWKFFDYDFGSDER, NGVLFLGLVGNSGIACVNEHQVLQR, DPQYEENNVQYEGSQDILNTQSFGK, IMENLPQSGRINDPEGNEYMLALSNR QNIDVVAR, NEYLLALSDR, QAAIQSGEYDR, QVEIPHDVATTGK, NEDTLQMVVSMK, SENQGNDVQYER, QAAIQSGEYDRTK, LLQPYPDWSFAKYEDCSGIVSAHK, NQNVLNNDLNLEHVNFQILGANVNDLIR, VYGMALSPVTHNLYYNSPSSENLYYVNTESLMK, NEYLLALSDRNQNVLNNDLNLEHVNFQILGANVNDLIR EYILVLSNK, IMNANVNELILNTR, MVNNDFNFDDVNFR, TVAQSDETLQMIASMK, VGDGGPLLQPYPDWSFAK, KVGDGGPLLQPYPDWSFAK, SLPILHEWKFFDYDFGSDER, TSDYQQNDIHYEGVQNILDTQSSAK LTSNTFDYDPR, IVNDDFNFDDVNFR


a Identified proteins from RJ of A. mellifera L. were categorized into fractions. The table lists peptide, score, accession no, protein names, theoritical pI, theoritical mass (kDa), species, functional category and matched amino acid sequence. Polymorphism of the MRJP 3 protein is a consequence of polymorphism of a region with a variable number of tandem repeats located at the C-terminal part of the MRJP3 coding region. So we regard these proteins are same MRJP3. The protein includes these proteins, which were idenified as major royal jelly protein 3 (gi|58585142) and royal jelly protein RJP57-1 (gi|1113119) (See Supplementary Figure 1).

88

405

15

2

604

54

339

833

81

9

score

peptide

Table 2. Continued


research articles


3210



2

3

protein name


1

spot no.

spieces


A. mellifera

A. mellifera

A. mellifera

5.10/48886.09

5.10/48886.09

5.10/48886.09

pI/MW (Da) theoretical acc. no.

gi|58585098

gi|58585098

gi|58585098

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

78

127

68

peptide

1483

1571

1321

score

67

65

66

sequence coverage (%)

SLPILHEWK, FFDYDFGSDERR, RQDAILSGEYDYK, QDAILSGEYDYK, NNYPSDIDQWHDK, IFVTMLR, YNGVPSSLNVISK, KVGDGGPLLQPYPDWSFAK, VGDGGPLLQPYPDWSFAK, YDDCSGIVSASK, YDDCSGIVSASKLAIDK, LAIDK, LAIDKCDR, LLTFDLTTSQLLK, LSSLAVQSLDCNTNSDTMVYIADEK, LTSNTFDYDPK, LTSNTFDYDPKFTK, TSDYQQNDIHYEGVQNILDTQSSAK, SGVLFFGLVGDSALGCWNEHR, TVAQSDETLQMIASMK, IKEALPHVPIFDR, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL SLPILHEWK, FFDYDFGSDER, FFDYDFGSDERR, RQDAILSGEYDYK, QDAILSGEYDYK, NNYPSDIDQWHDK, IFVTMLR, YNGVPSSLNVISK, KVGDGGPLLQPYPDWSFAK, VGDGGPLLQPYPDWSFAK, YDDCSGIVSASK, LAIDK, LAIDKCDR, LLTFDLTTSQLLK, GEGLIVYHNSDDSFHR, LTSNTFDYDPK, LTSNTFDYDPKFTK, TSDYQQNDIHYEGVQNILDTQSSAK, VVSKSGVLFFGLVGDSALGCWNEHR, SGVLFFGLVGDSALGCWNEHR, TVAQSDETLQMIASMK, IKEALPHVPIFDR, EALPHVPIFDR, EYILVLSNK, MQKMVNNDFNFDDVNFR, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL SLPILHEWK, FFDYDFGSDER, FFDYDFGSDERR, RQDAILSGEYDYK, QDAILSGEYDYK, NNYPSDIDQWHDK, IFVTMLR, KVGDGGPLLQPYPDWSFAK, VGDGGPLLQPYPDWSFAK, YDDCSGIVSASK, LAIDK, LAIDKCDR, LLTFDLTTSQLLK, LSSLAVQSLDCNTNSDTMVYIADEK, GEGLIVYHNSDDSFHR, LTSNTFDYDPK, LTSNTFDYDPKFTK, TSDYQQNDIHYEGVQNILDTQSSAK, SGVLFFGLVGDSALGCWNEHR, TVAQSDETLQMIASMK, IKEALPHVPIFDR, EALPHVPIFDR, EYILVLSNK, MQKMVNNDFNFDDVNFR, MVNNDFNFDDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL


Table 3. Royal Jelly Proteins (1st collection at 48 h; wild cherry and dandelion, etc., were blooming) of A. mellifera Separated by 2-DGE and Their Identification with nESI- LC-MS/ MSa




8


6

7


5

protein name


4

spot no.

Table 3. Continued

spieces

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09


gi|58585098

gi|58585098

gi|58585098

gi|58585098

gi|58585098

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

31

47

66

82

70

peptide

1117

1001

1162

1218

1353

score

56

48

55

55

65

sequence coverage (%) matched amino acid sequence

SLPILHEWK, FFDYDFGSDERR, RQDAILSGEYDYK, QDAILSGEYDYK, NNYPSDIDQWHDK, IFVTMLR, YNGVPSSLNVISK, KVGDGGPLLQPYPDWSFAK, VGDGGPLLQPYPDWSFAK, YD DCSGIVSASK, LAIDK, LAIDKCDR, LLTFDLTTSQLLK, LSSLAVQSLDCNTNSDTMVYIADEK, LTSNTFDYDPK, TSDYQQNDIHYEGVQNILDTQSSAK, SGVLFFGLVGDSALGCWNEHR, TVAQSDETLQMIASMK, IKEALPHVPIFDR, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL SLPILHEWK, FFDYDFGSDER, FFDYDFGSDERR, RQDAILSGEYDYK, QDAILSGEYDYK, NNYPSDIDQWHDK, IFVTMLR, KVGDGGPLLQPYPDWSFAK, VGDGGPLLQPYPDWSFAK, YDDC SGIVSASK, LAIDK, LAIDKCDR, LLTFDLTTSQLLK, GEGLIVYHNSDDSFHR, LTSNTFDYDPK, TSDYQQNDIHYEGVQNILDTQSSAK, TVAQSDETLQMIASMK, IKEALPHVPIFDR, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL SLPILHEWK, FFDYDFGSDER, FFDYDFGSDERR, RQDAILSGEYDYK, QDAILSGEYDYK, NNYPSDIDQWHDK, IFVTMLR, YNGVPSSLNVISK, KVGDGGPLLQPYPDWSFAK, VGDGGPLLQPYPDWSFAK, YDDCSGIVSASK, LLTFDLTTSQLLK, GEGLIVYHNSDDSFHR, LTSNTFDYDPK, TSDYQQNDIHYEGVQNILDTQSSAK, TVAQSDETLQMIASMK, EALPHVPIFDR, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK GESLNKSLPILHEWK, SLPILHEWK, FFDYDFGSDERR, RQDAILSGEYDYK, QDAILSGEYDYK, NNYPSDIDQWHDK, IFVTMLR, KVGDGGPLLQPYPDWSFAK, VGDGGPLLQPYPDWSFAK, YDDCSGIVSASK, LAIDK, LTSNTFDYDPK, LTSNTFDYDPKFTK, TSDYQQNDIHYEGVQNILDTQSSAK, TVAQSDETLQMIASMK, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK SLPILHEWK, FFDYDFGSDERR, RQDAILSGEYDYK, QDAILSGEYDYK, NNYPSDIDQWHDK, NNYPSDIDQWHDKIFVTMLR, IFVTMLR, YDDCSGIVSASK, LAIDK, LAIDKCDR, LLTFD LTTSQLLK, LSSLAVQSLDCNTNSDTMVYIADEK, LTSNTFDYDPK, TSDYQQNDIHYEGVQNILDTQSSAK, SGVLFFGLVGDSALGCWNEHR, TVAQSDETLQMIASMK, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK


research articles


3212




Major Royal Jelly Protein 3b


11

12

14

15

protein name

9

spot no.

Table 3. Continued

Journal of Proteome Research • Vol. 7, No. 8, 2008 6.83/51073.52

6.87/65695.81

6.87/65695.81

A. mellifera

A. mellifera

6.83/51073.52

6.83/51073.52

A. mellifera

A. mellifera

A. mellifera

spieces

pI/MW (Da) theoretical

gi|56422035

gi|56422035

gi|58585108

gi|58585108

gi|58585108

acc. no.

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

85

83

30

56

75

peptide

1286

1053

929

1022

1134

score

49

42

45

48

52


SLNVIHEWK, YFDYDFGSEER, YFDYDFGSEERR, RQAAIQSGEYDHTK, QAAIQSGEYDHTK, NYPFDVDQWR, NYPFDVDQWRDK, DKTFVTILR, TFVTILR, YDGVPSTLNVISGK, LLKPYPDWSFAEFK, IVSAFK, IAIDKFDR, TVPVCAPK, LHVFDLK, LTSNTFDYDPR, MTIDGESFTLK, SQFGENNVQYQGSEDILNTQSLAK, NGVLFVGLVGNSAVGCWNEHQSLQR, QNLEMVAQNDR, TLQMIAGMK, EELPHFVGSNKPVK, DEYMLVLSNR, IVNDDFNFDDVNFR NLEKSLNVIHEWK, SLNVIHEWK, YFDYDFGSEER, YFDYDFGSEERR, RQAAIQSGEYDHTK, QAAIQSGEYDHTK, NYPFDVDQWR, DKTFVTILR, TFVTILR, YDGVPSTLNVISGK, LLK PYPDWSFAEFK, IVSAFK, IAIDKFDR, TVPVCAPK, LHVFDLK, GDALIVYQNADDSFHR, LTSNTFDYDPR, MTIDGESFTLK, SQFGENNVQYQGSEDILNTQSLAK, QNLEMVAQNDR, QNLEMVAQNDRTLQMIAGMK, TLQMIAGMK, DEYMLVLSNR, IVNDDFNFDDVNFR SLNVIHEWK, YFDYDFGSEER, YFDYDFGSEERR, RQAAIQSGEYDHTK, QAAIQSGEYDHTK, NYPFDVDQWR, DKTFVTILR, YDGVPSTLNVISGK, LLKPYPDWSFAEFK, IVSAFK, IAIDKFDR, TVPVCAPK, GDALIVYQNADDSFHR, LTSNTFDYDPR, MTIDGESFTLK, NGVLFVGLVGNSAVGCWNEHQSLQR, TLQMIAGMK, DEYMLVLSNR, IVNDDFNFDDVNFR, ILGANVK SANNLAHSMK, VIYEWK, HIDFDFGSDER, HIDFDFGSDERR, SGEFDHTK, NYPFDVDR, NYPFDVDRWR, DKTFVTIER, TFVTIER, GGPLLRPYPDWSFAK, YEDCSGIVSAFK, IAVDKFDR, LWVLDSGLVNNNQPMCSPK, LLTFDLK, TNTMVYIADEK, GEGLIMYQNSDDSFHR, LTSNTFDYDPR, LTVAGESFTVK, NGVLFLGLVGNSGIACVNEHQVLQR, IMENLPQSGR, INDPEGNEYMLALSNR, ILGANVDDLMR, ILGANVDDLMRNTR SANNLAHSMK, VIYEWK, HIDFDFGSDER, HIDFDFGSDERR, SGEFDHTK, NYPFDVDR, NYPFDVDRWR, DKTFVTIER, TFVTIER, NNGVPSSLNVVTNK, GGPLLRPYPDWSFAK, YEDCSGIVSAFK, IAVDKFDR, LWVLDSGLVNNNQPMCSPK, LLTFDLK, GEGLIMYQNSDDSFHR, LTSNTFDYDPR, YTKLTVAGESFTVK, LTVAGESFTVK, NGVLFLGLVGNSGIACVNEHQVLQR, ESFDVVAQNEETLQMIVSMK, IMENLPQSGR, INDPEGNEYMLALSNR, IINNDFNFNDVNFR, ILGANVDDLMR, ILGANVDDLMRNTR





21

24


19



18

22


protein name

17

spot no.

Table 3. Continued

6.87/65695.81

A. mellifera

A. mellifera

A. mellifera

5.10/48886.09

5.10/48886.09

5.10/48886.09

6.87/65695.81

A. mellifera

A. mellifera

6.87/65695.81

A. mellifera

spieces


gi|58585098

gi|58585098

gi|58585098

gi|56422035

gi|56422035

gi|56422035

acc. no.

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

25

125

97

11

10

24

peptide

901

1385

1431

522

483

988

score

48

62

64

24

20

44


SANNLAHSMK, VIYEWK, HIDFDFGSDER, HIDFDFGSDERR, SGEFDHTKNYPFDVDR, NYPFDVDR, NYPFDVDRWR, DKTFVTIER, TFVTIER, GGPLLRPYPDWSFAK, IAVDKFDR, LLTTFDLK, TNTMVYIADEK, GEGLIMYQNSDDSFHR, LTSNTFDYDPR, LTVAGESFTVK, DPQYEENNVQYEGSQDILNTQSFGK, NGVLFLGLVGNSGIACVNEHQVLQR, ESFDVVAQNEETLQMIVSMK, IMENLPQSGR, INDPEGNEYMLALSNR, IINNDFNFNDVNFR, ILGANVDDLMR, ILGANVDDLMRNTR HIDFDFGSDER, NYPFDVDRWR, YEDCSGIVSAFK, LLTFDLK, LTSNTFDYDPR, LTVAGESFTVK, ESFDVVAQNEETLQMIVSMK, INDPEGNEYMLALSNR, ILGANVDDLMR, ILGANVDDLMRNTR HIDFDFGSDER, GGPLLRPYPDWSFAK, YEDCSGIVSAFK, LTSNTFDYDPR, YTKLTVAGESFTVK, LTVAGESFTVK, NGVLFLGLVGNSGIACVNEHQVLQR, INDPEGNEYMLALSNR, ILGANVDDLMR SLPILHEWK, FFDYDFGSDER, RQDAILSGEYDYK, QDAILSGEYDYK, NNYPSDIDQWHDK, IFVTMLR, YNGVPSSLNVISK, KVGDGGPLLQPYPDWSFAK, VGDGGPLLQPYPDWSFAK, YD DCSGIVSASK, LAIDK, LAIDKCDR, LLTFDLTTSQLLK, GEGLIVYHNSDDSFHR, LTSNTFDYDPK, LTSNTFDYDPKFTK, TSDYQQNDIHYEGVQNILDTQSSAK, SGVLFFGLVGDSALGCWNEHR, TVAQSDETLQMIASMK, IKEALPHVPIFDR, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL SLPILHEWK, RQDAILSGEYDYK, QDAILSGEYDYK, NNYPSDIDQWHDK, NNYPSDIDQWHDKIFVTMLR, IFVTMLR, YNGVPSSLNVISK, VGDGGPLLQPYPDWSFAK, YDDCSGIVSASK, LAIDK, LAIDKCDR, LLTFDLTTSQLLK, GEGLIVYHNSDDSFHR, LTSNTFDYDPK, LTSNTFDYDPKFTK, TSDYQQNDIHYEGVQNILDTQSSAK, SGVLFFGLVGDSALGCWNEHR, SGVLFFGLVGDSALGCWNEHRTLER, TVAQSDETLQMIASMK, IKEALPHVPIFDR, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL SLPILHEWK, RQDAILSGEYDYK, QDAILSGEYDYK, IFVTMLR, YNGVPSSLNVISK, VGDGGPLLQPYPDWSFAK, YDDCSGIVSASK, LLTFDLTTSQLLK, LSSLAVQSLDCNTNSDTMVYIAD EK, LTSNTFDYDPK, TSDYQQNDIHYEGVQNILDTQSSAK, TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK



research articles


3214



PREDICTED: similar to Glucocerebrosidase precursor isoform 2 PREDICTED: similar to Glucocerebrosidase precursor isoform 2 Major Royal Jelly Protein 1

28

29

32

31



27

30


protein name

25

spot no.

Table 3. Continued

A. mellifera

5.89/52915.46

5.10/48886.09

5.19/57952.35

A. mellifera

A. mellifera

5.19/57952.35

5.10/48886.09

5.10/48886.09

5.10/48886.09

A. mellifera

A. mellifera

A. mellifera

A. mellifera

spieces


gi|58585170

gi|58585098

gi|66511551

gi|66511551

gi|58585098

gi|58585098

gi|58585098

acc. no.

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Region; O-Glycosyl hydrolase]

Unknown [Region; O-Glycosyl hydrolase]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

36

71

12

14

22

15

19

peptide

921

1279

588

554

914

749

749

score

36

58

23

24

40

34

41


RQDAILSGEYDYK, KVGDGGPLLQPYPDWSFAK, VGDGGPLLQPYPDWSFAK, LLTFDLTTSQLLK, LTSNTFDYDPK, TSDYQQNDIHYEGVQNILDTQSSAK, SGVLFFGLVGDSALGCWNEHR, TV AQSDETLQMIASMK, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK SLPILHEWK, RQDAILSGEYDYK, QDAILSGEYDYK, VGDGGPLLQPYPDWSFAK, LLTFDLTTSQLLK, LTSNTFDYDPK, SGVLFFGLVGDSALGCWNEHR, TVAQSDETLQMIASMK, IKEALPH VPIFDR, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR SLPILHEWK, RQDAILSGEYDYK, QDAILSGEYDYK, KVGDGGPLLQPYPDWSFAK, VGDGGPLLQPYPDWSFAK, YDDCSGIVSASK, LLTFDLTTSQLLK, LTSNTFDYDPK, SGVLFFGLVGDSALGCWNEHR, TVAQSDETLQMIASMK, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK GTFHWYVSSR, CQNDGSLTLNIDTSKR, NLSEATQDQLIR, AYFDPK, IPIGGTDFSTR, KAVELNPDLR, TEYYQTFANYILK, NLLMTEACEGSFPLEK, VVLGSWER, AAAFITPSNEIVVVAYNDNNEK GTFHWYVSSR, YQTILGFGGAFTDSAGMNIK, NLSEATQDQLIR, IPIGGTDFSTR, KAVELNPDLR, AVELNPDLR, TEYYQTFANYILK, FIEEYK, NLLMTEACEGSFPLEK, AAAFITPSNEIVVVAYNDNNEK SLPILHEWK, FFDYDFGSDER, RQDAILSGEYDYK, QDAILSGEYDYK, NNYPSDIDQWHDK, IFVTMLR, YNGVPSSLNVISK, KVGDGGPLLQPYPDWSFAK, VGDGGPLLQPYPDWSFAK, YD DCSGIVSASK, LAIDK, LAIDKCDR, LLTFDLTTSQLLK, GEGLIVYHNSDDSFHR, LTSNTFDYDPK, TSDYQQNDIHYEGVQNILDTQSSAK, TVAQSDETLQMIASMK, IKEALPHVPIFDR, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL YLDYDFDNDER, YLDYDFDNDERR, RQAAIQSGEYDR, QAAIQSGEYDR, QAAIQSGEYDRTK, TFLAVIR, YNGVPSSLNVVSDK, LLQPYPDWSFAK, YEDCSGIVSAHK, IAIDEYER, NTLIIYQNADDSFHR, LSSHTLNHNSDK, LSSHTLNHNSDKMSDQQENLTLK, MSDQQENLTLK, SENQGNDVQYER, QNIDVVAR, NEDTLQMVVSMK, IKQNVPQSGR, QNVPQSGR, NEYLLALSDR,





38

40


37



36

39


protein name

34

spot no.

Table 3. Continued

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

spieces

5.89/52915.46

5.89/52915.46

5.89/52915.46

5.89/52915.46

5.89/52915.46

5.89/52915.46


gi|58585170

gi|58585170

gi|58585170

gi|58585170

gi|58585170

gi|58585170

acc. no.

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

37

36

35

35

33

38

peptide

720

955

837

570

795

906

score

32

39

35

22

40

43


YLDYDFDNDERR, RQAAIQSGEYDR, QAAIQSGEYDR, QAAIQSGEYDRTK, TFLAVIR, YNGVPSSLNVVSDK, LLQPYPDWSFAK, YEDCSGIVSAHK, IAIDEYER, GELVSLTVQAMDSTN TMVYMVDNK, NTLIIYQNADDSFHR, LSSHTLNHNSDK, LSSHTLNHNSDKMSDQQENLTLK, SENQGNDVQYER, VQDVFDSQLTVK, QNIDVVAR, NEDTLQMVVSMK, IKQNVPQSGR, QNVPQSGR, NEYLLALSDR YLDYDFDNDER, YLDYDFDNDERR, QAAIQSGEYDR, TFLAVIR, YEDCSGIVSAHK, IAIDEYER, QVEIPHDVATTGK, NTLIIYQNADDSFHR, LSSHTLNHNSDK, LSSHTLNHNSDKMSD QQENLTLK, MSDQQENLTLK, SENQGNDVQYER, NGVLLFGLANNTLSCWNEHQSLDR, QNIDVVAR, NEDTLQMVVSMK, NEYLLALSDR, CANFDNQDNNHYNHNHNQAR YLDYDFDNDER, YLDYDFDNDERR, QAAIQSGEYDR, TFLAVIR, LLQPYPDWSFAK, IAIDEYER, LSSHTLNHNSDK, SENQGNDVQYER, QNIDVVAR, NEDTLQMVVSMK, NEYLLALSD R YLDYDFDNDER, YLDYDFDNDERR, RQAAIQSGEYDR, QAAIQSGEYDR, TFLAVIR, YNGVPSSLNVVSDK, LLQPYPDWSFAK, IAIDEYER, LFAFDLNTSQLLK, NTLIIYQNADDSFHR,LSSHTLNHNSDK, LSSHTLNHNSDKMSDQQENLTLK, MSDQQENLTLK, SENQGNDVQYER, QNIDVVAR, NEDTLQMVVSMK, IKQNVPQSGR, NEYLLALSDR YLDYDFDNDER, YLDYDFDNDERR, RQAAIQSGEYDR, QAAIQSGEYDR, QAAIQSGEYDRTK, TFLAVIR, YNGVPSSLNVVSDK, LLQPYPDWSFAK, IAIDEYER, LFAFDLNTSQLLK, NTLIIYQNADDSFHR, LSSHTLNHNSDK, MSDQQENLTLK, SENQGNDVQYER, VQDVFDSQLTVK, QNIDVVAR, NEDTLQMVVSMK, IKQNVPQSGR, NEYLLALSDR, YLDYDFDNDERR, QAAIQSGEYDR, TFLAVIR, YNGVPSSLNVVSDK, LLQPYPDWSFAK, IAIDEYER, LFAFDLNTSQLLK, LSSHTLNHNSDK, MSDQQENLTLK, SENQGNDVQYER, QHIDVVAR, NEDTLQMVVSMK, IKQNVPQSGR, NEYLLALSDR



research articles


3216



Major Royal Jelly Protein 1 Major Royal Jelly Protein 1 Major Royal Jelly Protein 1



PREDICTED: similar to Glucose Dehydrogenase isoform 1 Major Royal Jelly Protein 5

Glucose Oxidase

43

46

54

55

57

68

59

53

49


42

protein name


41

spot no.

Table 3. Continued

spieces

A. mellifera

A. mellifera

A. mellifera

6.48/67938.03

5.95/70235.97

5.58/69349.46

6.83/51073.52

6.87/65695.81

A. mellifera

A. mellifera

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera


gi|58585090

gi|58585138

gi|110756961

gi|58585108

gi|56422035

gi|58585098

gi|58585098

gi|58585098

gi|58585098

gi|58585098

gi|58585098


Unknown [Nutrition]

Unknown

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition] Unknown [Nutrition] Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

16

21

3

22

6

13

4

3

10

15

17

peptide

410

736

125

805

312

592

227

184

591

768

869

score

16

28

6

42

13

26

8

10

29

31

40


LANSMNVIHEWK, YLDYDFGSDER, NYPFDVDQWR, GMTFVTVPR, YKGVPSSLNVISEK, GVPSSLNVISEK, IAIDKFDR, LTSNTFDYDPK, SLYYVNTKPFMK, SEYGANNVQYQGVQDIFNTESIAK, ENMDMVAQNEETLQTVVAMK, VDTMDTMDTMDR, QMNEYMMALSMK, FINNDYNFNEVNFR SGGLMNVER, FPYQPPFAWK, AFITPFENR, IIFAR, MSALNVQPTSK, SVMIQAIR, DLGVEFQK, MGPSYDPMAVVSPR, VADASVQPQVISGNPVASVNMVGER

RQDAILSGEYDYK, VGDGGPLLQPYPDWSFAK, YDDCSGIVSASK, LAIDKCDR, LLTFDLTTSQLLK, LTSNTFDYDPK, SGVLFFGLVGDSALGCWNEHR, TVAQSDETLQMIASMK, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL RQDAILSGEYDYK, QDAILSGEYDYK, VGDGGPLLQPYPDWSFAK, YDDCSGIVSASK, LLTFDLTTSQLLK, LTSNTFDYDPK, TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL RQDAILSGEYDYK, YDDCSGIVSASK, TSDYQQNDIHYEGVQNILDTQSSAK, TVAQSDETLQMIASMK, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK VGDGGPLLQPYPDWSFAK, LLTFDLTTSQLLK, IMNANVNELILNTR LLTFDLTTSQLLK, LTSNTFDYDPK, IMNANVNELILNTR QDAILSGEYDYK, LAIDKCDR, LLTFDLTTSQLLK, LTSNTFDYDPK, TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, CENPDNDRTPFK, ISIHL TNTMVYIADEK, LTSNTFDYDPR, INDPEGNEYMLALSNR, IINNDFNFNDVNFR, ILGANVDDLMR SLNVIHEWK, YFDYDFGSEER, YFDYDFGSEERR, RQAAIQSGEYDHTK, QAAIQSGEYDHTK, TFVTILR, YDGVPSTLNVISGK, IVSAFK, IAIDKFDR, GDALIVYQNADDSFHR, LTSNTFDYDPR, MTIDGESFTLK, SQFGENNVQYQGSEDILNTQSLAK, QNLEMVAQNDR, TLQMIAGMK, IVNDDFNFDDVNFR, NTHCVNNNQNDNIQNTNNQNDNNQK KYHSTGGLLNVER, STATAFLRPFR, VTSGNTAAPAIMIGER




Glucose Oxidase





71

74

76

82

83

84

protein name

61

spot no.

Table 3. Continued

6.87/65695.81

6.87/65695.81

A. mellifera

6.87/65695.81

A. mellifera

A. mellifera

5.95/70235.97

6.48/67938.03

5.95/70235.97

5.95/70235.97

A. mellifera

A. mellifera

A. mellifera

A. mellifera

spieces


gi|56422035

gi|56422035

gi|56422035

gi|58585138

gi|58585090

gi|58585138

gi|58585138

acc. no.

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]


Unknown [Nutrition]

Unknown [Nutrition]

functional category

32

17

30

31

24

24

30

peptide

1004

622

892

615

680

787

777

score

35

27

32

22

26

30

28


LANSMNVIHEWK, YLDYDFGSDER, YLDYDFGSDERR, NYPFDVDQWR, GMTFVTVPR, YKGVPSSLNVISEK, GVPSSLNVISEK, LLQPYPDWSWANYK, DCSGIVSAYK, IAIDKFDR, LTSNTFDYDPK, SLYYVNTKPFMK, ENMDMVAQNEETLQTVVAMK, MDRVDTMDTMDTMDR, QMNEYMMALSMK, FINNDYNFNEVNFR YLDYDFGSDER, YLDYDFGSDERR, NYPFDVDQWR, GMTFVTVPR, YKGVPSSLNVISEK, GVPSSLNVISEK, LLQPYPDWSWANYK, DCSGIVSAYK, IAIDKFDR, LHVFDLNTSHQLK, LTSNTFDYDPK, SLYYVNTKPFMK, ENMDMVAQNEETLQTVVAMK, MMHLPQSNK, VDTMDTMDTMDRMDR, QMNEYMMALSMK, FINNDYNFNEVNFR LSEVSNWK, NLGGTTLHHGMAYHR, SGGLMNVER, FPYQPPFAWK, AFITPFENR, RATGVNVLINGR, EVILSAGSVNTPQLLMLSGIGPK, IADLSAHDK, IADLSAHDKQAVR, MSALNVQPTSK, SVMIQAIR, DLGVEFQK, MGPSYDPMAVVSPR, VADASVQPQVISGNPVASVNMVGER YLDYDFGSDER, YLDYDFGSDERR, NYPFDVDQWR, GMTFVTVPR, LLQPYPDWSWANYK, DCSGIVSAYK, IAIDKFDR, LTSNTFDYDPK, SLYYVNTKPFMK, ENMDMVAQNEETLQTVVAMK, MHTMDTMDTMDR, FINNDYNFNEVNFR SANNLAHSMK, VIYEWK, HIDFDFGSDER, NYPFDVDRWR, DKTFVTIER, TFVTIER, NNGVPSSLNVVTNK, YEDCSGIVSAFK, IAVDKFDR, IAVDKFDR, LWVLDSGLVNNNQPMCSPK,TNTMVYIADEK, GEGLIMYQNSDDSFHR, LTVAGESFTVK, IMENLPQSGR, INDPEGNEYMLALSNR, ILGANVDDLMR, ILGANVDDLMRNTR SANNLAHSMK, NYPFDVDR, GGPLLRPYPDWSFAK, YEDCSGIVSAFK, IAVDKFDR, LWVLDSGLVNNNQPMCSPK, LLTFDLK, TNTMVYIADEK, LTSNTFDYDPR, LTVAGESFTVK, IMENLPQSGR, INDPEGNEYMLALSNR, ILGANVDDLMR SANNLAHSMK, VIYEWK, HIDFDFGSDER, NYPFDVDR, DKTFVTIER, TFVTIER, NNGVPSSLNVVTNK, YEDCSGIVSAFK, IAVDKFDR, LWVLDSGLVNNNQPMCSPK, LLTFDLK, TNTMVYIADEK, LTVAGESFTVK, DPQYEENNVQYEGSQDILNTQSFGK, IMENLPQSGR, INDPEGNEYMLALSNR, IINNDFNFNDVNFR, ILGANVDDLMR



research articles


3218







90

92

94

96

99

95


88

protein name


86

spot no.

Table 3. Continued

spieces

5.10/48886.09

6.87/65695.81

A. mellifera

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09

6.87/65695.81

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera


gi|56422035

gi|58585098

gi|58585098

gi|58585098

gi|58585098

gi|58585098

gi|58585098

gi|56422035

Unknown [Nutrition]

Unknown [Nutrition]


Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

16

23

28

4

5

5

5

44

peptide

620

822

1090

258

282

335

328

967

score

27

42

48

12

14

18

14

34



KSANNLAHSMK, SANNLAHSMK, VIYEWK, HIDFDFGSDER, HIDFDFGSDERR, SGEFDHTK, NYPFDVDRWR, DKTFVTIER, TFVTIER, NNGVPSSLNVVTNK, YEDCSGIVSAFK, IAVDKFDR, LWVLDSGLVNNNQPMCSPK, LLTFDLK, TNTMVYIADEK, LTSNTFDYDPR, LTVAGESFTVK, IMENLPQSGR, INDPEGNEYMLALSNR, ILGANVDDLMR, YDDCSGIVSASK, LLTFDLTTSQLLK, LTSNTFDYDPK, MVNNDFNFDDVNFR, IMNANVNELILNTR LTSNTFDYDPK, TSDYQQNDIHYEGVQNILDTQSSAK, TVAQSDETLQMIASMK, MVNNDFNFDDVNFR, IMNANVNELILNTR YDDCSGIVSASK, LTSNTFDYDPK, TVAQSDETLQMIASMK, EALPHVPIFDR, IMNANVNELILNTR TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR SLPILHEWK, RQDAILSGEYDYK, QDAILSGEYDYK, VGDGGPLLQPYPDWSFAK, YDDCSGIVSASK, LAIDKCDR, LLTFDLTTSQLLK, GEGLIVYHNSDDSFHR, LTSNTFDYDPK, LTSNTFDYDPKFTK, TSDYQQNDIHYEGVQNILDTQSSAK, TVAQSDETLQMIASMK, IKEALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL RQDAILSGEYDYK, LLTFDLTTSQLLK, LSSLAVQSLDCNTNSDTMVYIADEK, GEGLIVYHNSDDSFHR, LTSNTFDYDPK, LTSNTFDYDPKFTK, TSDYQQNDIHYEGVQNILDTQSSAK, TVAQSDETLQMIASMK, IKEALPHVPIFDR, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK SANNLAHSMK, SGEFDHTK, SGEFDHTKNYPFDVDR, NYPFDVDR, NYPFDVDRWR, DKTFVTIER, GGPLLRPYPDWSFAK, YEDCSGIVSAFK, IAVDKFDR, YTKLTVAGESFTVK, LTVAGESFTVK, DPQYEENNVQYEGSQDILNTQSFGK, ESFDVVAQNEETLQMIVSMK, IMENLPQSGR, INDPEGNEYMLALSNR


Vicilin

Major Royal Jelly Protein 5 Major Royal Jelly Protein 3b Major Royal Jelly Protein 2

102

103

113


PREDICTED: similar to Ferritin 2 Light Hhain homologue CG1469-PA, isoform A Major Royal Jelly Protein 2

110

111


109

108

104


101

protein name


100

spot no.

Table 3. Continued

A. mellifera

5.10/48886.09

6.83/51073.52

6.10/25185.74

A. mellifera

A. mellifera

6.83/51073.52

A. mellifera

6.83/51073.52

6.87/65695.81

A. mellifera

A. mellifera

5.95/70235.97

A. mellifera

5.39/52231.43

6.87/65695.81

A. mellifera

Pisum sativum

6.87/65695.81

spieces

A. mellifera


gi|58585098

gi|58585108

gi|66524161

gi|58585108

gi|58585108

gi|56422035

gi|58585138

gi|313671

gi|56422035

gi|56422035

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Region; Eukaryotic Ferritin]

Unknown [Nutrition]


Unkown [region; Cupin_1]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

7

20

3

11

17

8

4

13

10

36

peptide

424

350

113

37

539

136

149

379

466

1038

score

20

17

17

8

23

8

5

23

21

42



LTSNTFDYDPR, SQFGENNVQYQGSEDILNTQSLAK, QNLEMVAQNDR, QNLEMVAQNDRTLQMIAGMK, TLQMIAGMK, DEYMLVLSNR, IVNDDFNFDDVNFR YNGVPSSLNVISK, LTSNTFDYDPK, TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK

YEDCSGIVSAFK, LTSNTFDYDPR, INDPEGNEYMLALSNR SLNVIHEWK, YFDYDFGSEER, YFDYDFGSEERR, RQAAIQSGEYDHTK, NYPFDVDQWR, DKTFVTILR, TFVTILR, YDGVPSTLNVISGK, IVSAFK, LHVFDLK, GDALIVYQNADDSFHR, MTIDGESFTLK TFVTILR, LHVFDLK, DKTFVTILR, MTIDGESFTLK, YFDYDFGSEER, QAAIQSGEYDHTK, YDGVPSTLNVISGK, YFDYDFGSEERR GGSMNFGQEPK, KQDSAIAHYMEEK, FLLMSTYLGNYENQR

SANNLAHSMK, VIYEWK, HIDFDFGSDER, DAAIK, SGEFDHTKNYPFDVDR, NYPFDVDR, NYPFDVDRWR, DKTFVTIER, TFVTIER, NNGVPSSLNVVTNK, GGPLLRPYPDWSFAK, YEDCSGIVSAFK, IAVDKFDR, LWVLDSGLVNNNQPMCSPK, LLTFDLK, TNTMVYIADEK, GEGLIMYQNSDDSFHR, LTSNTFDYDPR, YTKLTVAGESFTVK, LTVAGESFTVK, DPQYEENNVQYEGSQDILNTQSFGK, IMENLPQSGR, INDPEGNEYMLALSNR, ILGANVDDLMR SANNLAHSMK, YEDCSGIVSAFK, IAVDKFDR, LWVLDSGLVNNNQPMCSPK, LTSNTFDYDPR, LTVAGESFTVK, DPQYEENNVQYEGSQDILNTQSFGK, IMENLPQSGR, INDPEGNEYMLALSNR FQTLFENENGHIR, SKIFENLQNYR, IFENLQNYR, LLEYK, AILTVLKPDDR, SVSSESEPFNLR, FFEITPEK, EGSLLLPHYNSR, ASSNLDLLGFGINAENNQR, ELAFPGSAQEVDR, ERGSR YLDYDFGSDER, GMTFVTVPR, SLYYVNTKPFMK


research articles


3220



PREDICTED: similar to Ferritin 1 Heavy Chain homologue CG2216-PE, isoform E Major Royal Jelly Protein 1 Major Royal Jelly Protein 1 Major Royal Jelly Protein 1






116

118


121

125

131

132

133

134

130

126

124

123

122

120

119

117


115

protein name


114

spot no.

Table 3. Continued

spieces

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09

6.83/51073.52

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.79/17351.39

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09

5.10/48886.09


gi|58585098

gi|58585098

gi|58585098

gi|58585098

gi|58585098

gi|58585108

gi|58585098

gi|58585098

gi|58585098

gi|58585098

gi|110762641

gi|58585098

gi|58585098

gi|58585098

gi|58585098

gi|58585098

gi|58585098

gi|58585098

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]



Unknown [Region; Eukaryotic Ferritin]



Unknown [Nutrition]

Unknown [Nutrition]

functional category

8

8

6

5

6

8

5

11

7

4

4

19

7

5

5

6

14

6

peptide

381

408

308

315

196

111

278

365

254

191

195

345

214

229

288

265

481

453

score

14

17

14

14

13

5

12

16

12

9

21

16

10

12

13

11

27

17


TVAQSDETLQMIASMK, EYILVLSNK, IMNANVNELILNTR TVAQSDETLQMIASMK, EALPHVPIFDR, EYILVLSNK, IMNANVNELILNTR, ISIHL TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR RQAAIQSGEYDHTK, QAAIQSGEYDHTK, IVSAFK, IAIDK TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, ISIHL TVAQSDETLQMIASMK, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR TVAQSDETLQMIASMK, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR LLTFDLTTSQLLK, LTSNTFDYDPK, TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR LTSNTFDYDPK, TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR

LLTFDLTTSQLLK, LTSNTFDYDPK, TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR LTSNTFDYDPK, TSDYQQNDIHYEGVQNILDTQSSAK, TVAQSDETLQMIASMK, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL QDAILSGEYDYK, VGDGGPLLQPYPDWSFAK, YDDCSGIVSASK, LAIDK, LAIDKCDR LTSNTFDYDPK, TVAQSDETLQMIASMK, MVNNDFNFDDVNFR, IMNANVNELILNTR, ISIHL TVAQSDETLQMIASMK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK TVAQSDETLQMIASMK, EYILVLSNK, IMNANVNELILNTR, ISIHL TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, CENPDNDRTPFK, ISIHL VLEYLLMR, SLESQVNVEIK, GELTNINEDNVLLR


Glucose Oxidase

Glucose Oxidase

PREDICTED: similar to Ferritin 2 Light Chain homologue CG1469-PA, isoform A Major Royal Jelly Protein 2 Albumin



PREDICTED: similar to Ferritin 1 Heavy Chain homologue CG2216-PE, isoform E Major Royal Jelly Protein 1 Odorant Binding Protein 14 Major Royal Jelly Protein 1 Major Royal Jelly Protein 1 Major Royal Jelly Protein 7

136

137

138

141

142

143

P1

147

146

145

144

140

139


protein name

135

spot no.

Table 3. Continued

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

Bos taurus

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

spieces

4.90/50540.92

5.10/48886.09

5.10/48886.09

5.71/15200.74

5.10/48886.09

5.79/17351.39

5.10/48886.09

6.83/51073.52

5.82/69293.41

6.83/51073.52

6.10/25185.74

6.48/67938.03

6.48/67938.03

5.10/48886.09


gi|62198227

gi|58585098

gi|58585098

gi|94158822

gi|58585098

gi|110762641

gi|58585098

gi|58585108

gi|62648

gi|58585108

gi|66524161

gi|58585090

gi|58585090

gi|58585098

acc. no.


Unknown [Nutrition] Unknown

Unknown

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition] Binding

Carbohydrate Metabolism Carbohydrate Metabolism Unknown [Region; Eukaryotic Ferritin]

Unknown [Nutrition]

functional category

28

9

3

6

6

2

8

3

14

7

3

11

5

13

peptide

988

328

198

77

158

106

362

170

495

138

87

137

142

349

score

52

12

6

13

9

14

15

10

17

7

13

7

7

18


TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR VMHEWK, YIDYDFGSEEKR, QAAIQSDEYDHTK, NYPFDVDQWR, DKTFVTVLR, TFVTVLR, YDGVPSSLNVISEK, LLQPYPDWSWTK, DCSGIVSAYSIAIDK, DCSGIVSAYSIAIDKFDR, LLVFDLNSSQLIK, QVDIPHEIAVNTTTEQGR, SLAVQAISSVNTLVYIADNK, GDGLIVYQNSDDSFHR, VQYNGVQDVFNTQTTAK, ENTDMVAQNEETLQMIVGMK, ILNNDLNFNDINFR, ITIDASFN

MVNNDFNFDDVNFR, IMNANVNELILNTR

TVAQSDETLQMIASMK, EALPHVPIFDR, EYILVLSNK, ISIHL AVMELLIDENSVK, SVDFL

YFDYDFGSEER, YDGVPSTLNVISGK, LTSNTFDYDPR YLYEIAR, KQTALVELLK, LVNELTEFAK, HLVDEPQNLIK, TVMENFVAFVDK, RHPEYAVSVLLR, LGEYGFQNALIVR, DAFLGSFLYEYSR, KVPQVSTPTLVEVSR LTSNTFDYDPR, SQFGENNVQYQGSEDILNTQSLAK, QNLEMVAQNDR LTSNTFDYDPK, TVAQSDETLQMIASMK, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, ISIHL VLEYLLMR, GELTNINEDNVLLR

LTSNTFDYDPK, TVAQSDETLQMIASMK, EALPHVPIFDR, EYILVLSNK, MVNNDFNFDDVNFR, IMNANVNELILNTR, ISIHL IADLSAHDK, MGPSYDPMAVVSPR, VADASVQPQVISGNPVASVNMVGER IADLSAHDK, MGPSYDPMAVVSPR, VADASVQPQVISGNPVASVNMVGER FLLMSTYLGNYENQR, YSDEMWENGIDLIK



research articles


3222






P3

P4

P5

P6

A. mellifera

A. mellifera

A. mellifera

A. mellifera

A. mellifera

spieces

6.83/51073.52

6.83/51073.52

4.90/50540.92

4.90/50540.92

4.90/50540.92


gi|58585108

gi|58585108

gi|62198227

gi|62198227

gi|62198227

acc. no.

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

Unknown [Nutrition]

functional category

86

75

30

32

41

peptide

1156

1096

810

971

1202

score

55

56

41

56

56


VMHEWK, YIDYDFGSEEKR, RQAAIQSDEYDHTK, QAAIQSDEYDHTK, NYPFDVDQWR, NYPFDVDQWRDK, TFVTVLR, YDGVPSSLNVISEK, LLQPYPDWSWTK, DCSGIVSAYSIAIDK, DCSGIVSAYSIAIDKFDR, LLVFDLNSSQLIK, SLAVQAISSVNTLVYIADNK, GDGLIVYQNSDDSFHR, LTSNTFNYDPR, DLYYVNTKPFIK, VQYNGVQDVFNTQTTAK, ENTDMVAQNEETLQMIVGMK, ILNNDLNFNDINFR, ILIGGVSDLLENTR, CTNFNIQNDDSDENNDDSIR, VMHEWK, YIDYDFGSEEKR, QAAIQSDEYDHTK, NYPFDVDQWR, DKTFVTVLR, TFVTVLR, YDGVPSSLNVISEK, LLQPYPDWSWTK, DCSGIVSAYSIAIDK, DCSGIVSAYSIAIDKFDR, QVDIPHEIAVNTTTEQGR, SLAVQAISSVNTLVYIADNK, GDGLIVYQNSDDSFHR, LTSNTFNYDPR, DLYYVNTKPFIK, VQYNGVQDVFNTQTTAK, ENTDMVAQNEETLQMIVGMK, ILNNDLNFNDINFR, CTNFNIQNDDSDENNDDSIR, ITIDASFN VMHEWK, YIDYDFGSEEKR, RQAAIQSDEYDHTK, QAAIQSDEYDHTK, NYPFDVDQWR, TFVTVLR, YDGVPSSLNVISEK, DCSGIVSAYSIAIDK, QVDIPHEIAVNTTTEQGR, LTSNTFNYDPR, DLYYVNTKPFIK, ENTDMVAQNEETLQMIVGMK, ILNNDLNFNDINFR, ILIGGVSDLLENTR, CTNFNIQNDDSDENNDDSIR SLNVIHEWK, YFDYDFGSEER, QAAIQSGEYDHTK, NYPFDVDQWR, DKTFVTILR, TFVTILR, YDGVPSTLNVISGK, LLKPYPDWSFAEFK, IVSAFK, IAIDK, IAIDKFDR, TVPVCAPK, LHVFDLK, GDALIVYQNADDSFHR, LTSNTFDYDPR, MTIDGESFTLK, SQFGENNVQYQGSEDILNTQSLAK, QNLEMVAQNDR, TLQMIAGMK, IKEELPHFVGSNKPVK, EELPHFVGSNKPVK, IVNDDFNFDDVNFR, ILGANVK, NTHCVNNNQNDNIQNTNNQNDNNQK SLNVIHEWK, YFDYDFGSEER, YFDYDFGSEERR, RQAAIQSGEYDHTK, QAAIQSGEYDHTK, NYPFDVDQWR, DKTFVTILR, YDGVPSTLNVISGK, LLKPYPDWSFAEFK, DCSKIVSAFK, IVSAFK, IAIDK, IAIDKFDR, LHVFDLK, GDALIVYQNADDSFHR, LTSNTFDYDPR, MTIDGESFTLK, SQFGENNVQYQGSEDILNTQSLAK, QNLEMVAQNDR, TLQMIAGMK, IKEELPHFVGSNKPVK, EELPHFVGSNKPVK, IVNDDFNFDDVNFR, ILGANVK, NTHCVNNNQNDNIQNTNNQNDNNQK


a Identified proteins from RJ of A. mellifera L. were categorized into fractions. The table lisits peptide, protein names, species, theoritical pI, theoritical mass (kDa), accession no, functional category, score, sequence coverage (%), and matched amino acid sequence. b The protein was identified as royal jelly protein RJP57-1(gi|1113119), but from sequence similality (100%) with MRJP 3, we detected the protein is MRJP 3 (See Supplementary Figure 1).


protein name

P2

spot no.

Table 3. Continued


research articles

RJ Proteomics Reveals Novel RJ Proteins and Phospho/Glycoproteins Table 4. Total Nonredundant Identified Proteins from 1-DGE/2-DGE and MS Analysis acc. no

gi|58585098 gi|58585108 gi|56422035 gi|58585170 gi|58585138 gi|42601246 gi|62198227 gi|60115688 gi|6448461 gi|4433771 gi|23126818 gi|4574723 gi|33562986 gi|15620867 gi|23271256 gi|71738784 gi|17509865 gi|189036 gi|1346640 gi|6981236 gi|21703262 gi|31979 gi|32450747 gi|19527172 gi|71546273 gi|8568805 gi|409450 gi|8925552 gi|5738567 gi|14558062 gi|58585164 gi|9931616 gi|313671 gi|94158822 gi|162648 gi|14961780 gi|763431 gi|114669123 gi|66547819 gi|11077642 gi|11808552 gi|89045073 gi|48118838 gi|66511551 gi|110756961 gi|110762641 gi|66524161 gi|66557660 gi|48094573 gi|47228503 gi|2627260 gi|29888

protein name

1D 1-48

Major Royal Jelly Protein 1 Major Royal Jelly Protein 2b Major Royal Jelly Protein 3c Major Royal Jelly Protein 4 Major Royal Jelly Protein 5 Major Royal Jelly Protein MRJP5 precursor Major Royal Jelly Protein 7b Venom Protein 2 precursor Glucose Oxidase MHC class II beta 1 COG1020: Non-Ribosomal Peptide Synthetase modules and related proteins putative Replication Protein E1 ORF18 KIAA1904 protein Gametogenetin ACP54A1 Y37E3.8b Nonmuscle Myosin Heavy Chain (NMHC) Myosin-10 Myosin, Heavy Polypeptide 9 Cytoplasmic Actin 2 Histone H2A.2 Anxal-Prov Protein (Annexin A1) Smooth Muscle Cell Associated Protein-1 ABC Transporter putative ABC Transporter, ATP-Binding Acetyl-CoA Carboxylase Light-Harvesting complex Protein LHCG4 POL Protein E2F Transcription Factor 7 Alpha-Glucosidase sof2460 precursor Vicilin Odorant Binding Protein 14 Albumin Histone H2B isoform 2 Human Albumin myosin, heavy polypeptide 10, nonmuscle isoform 1 Major Royal Jelly Protein MRJP5 Yellow-h CG1629-PA, partial Voltage-Gated Sodium Channel Alpha Kinase Suppressor of Ras-1 (Kinase suppressor of ras) Y4C6B.6 Glucocerebrosidase precursor isoform 2 Glucose Dehydrogenase isoform 1 Ferritin 1 Heavy Chain homologue CG2216-PE, isoform E Ferritin 2 Light Chain homologue CG1469-PA, isoform A Hypothetical Protein Hypothetical Protein unnamed protein product unnamed protein product unnamed protein product

b b b b b b b b b b b b

1D 2-48a

b b b b b

2-DGE

b b b b b b b

b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b

b

b b b b b

b b b b b

a Data from only the two differentially exppressed bands in the second collection/48 h. b Identified as a phosphoprotein. c Polymorphism of the MRJP 3 protein is a consequence of polymorphism of a region with a variable number of tandem repeats located at the C-terminal part of the MRJP3 coding region. So we regard these proteins are same MRJP3. The protein includes these proteins, which were idenified as major royal jelly protein 3 (gi|58585142) and royal jelly protein RJP57-1 (gi|1113119) (See Supplementary Figure 1). Predicted proteins are indicated in italics.

for MS analysis. Results of the MS identifications are presented in Tables 1 (RJ 1-48) and 2 (RJ 2-48). 3.2.1. The Identified RJ Proteins by 1-DGE Analysis. In the RJ 1-48 sample used for 1-D MS analysis, a total of 90 proteins were identified from the 14 excised fractions representing 44 nonredundant proteins (Tables 1 and 4), the fractions 1-14, and gave protein assignments of 8, 16, 13, 7, 9, 3, 3, 6, 5, 3, 5, 4, 5 and 3 proteins, respectively. On the other hand, RJ proteins

from fraction numbers 2-4 and 2-5 resulted in the identification of 9 and 6 proteins, respectively (Tables 2 and 4). Out of these 47 nonredundant proteins, 37 proteins were identified for the first time from RJ. We discuss below these identified protein families/proteins. 3.2.1.1. Major Royal Jelly Protein (MRJP) Family. The MRJPs constitute about 90% of total RJ protein.9,15 Nine A. mellifera loci encoding MRJPs (MRJP 1 to MRJP 9) have been Journal of Proteome Research • Vol. 7, No. 8, 2008 3223

research articles

Furusawa et al.

1,16

identified, but little is known about the function of these genes or their protein products. It has been previously reported that the MRJP 1 gene is expressed in the mushroom bodies of the honey bee brain, implicating this gene in behavior.17 Therefore, it can be suggested that the MRJPs can be multifunctional, performing a nutritional role as a component of RJ and executing additional roles in various tissues of the honey bee including the brain. However, it should be noted that the expression patterns of the MRJP genes across development and in different sexes and castes have not been well-characterized16 (for additional information see ref 18). We could identify MRJP 1, 2, 3, 4, and 5, but on could not identify MRJP 6, 7, 8, and 9 by 1-DGE analysis. Previously, only one report could identify MRJP 6, 7, and 9.10 No MRJP 8 has been detected to date from RJ; however, the protein was identified in the proteome complement of the secretion from the hypopharyngeal gland of Africanized nurse honeybees.15 Thus, the MRJP 8 from RJ is yet to be identified. MRJP family components have high similarity (see Supplementary Figure 2), which might be one reason we have not been able to identify the MRJP 8 member. 3.2.1.2. Glucose Oxidase and Venom Protein 2. Two proteins, glucose oxidase and venom protein 2, were identified. These have also been identified previously.10,11 The glucose oxidase protein is of interest in relation to its antibacterial properties in honey. It catalyzes glucose to form gluconic acid and hydrogen peroxide (H2O2), the main agent responsible for antibacterial activity in most honeys; thus, the enzyme is thought to help in the degradation of the RJ components for larvae feeding and in defense.18 The venom protein 2 is found in honey bee hypopharyngeal grand and it has a plateletderived and vascular endothelial growth factor family domain; but the protein shows no homologies with any known protein.19 3.2.1.3. Novel Proteins. A total of 37 novel proteins were identified, and although the functions of many of these proteins are unknown, we discuss briefly some of them based on the information available in public databases. Anxa1-Prov protein (Annexin A1), a member of the annexin family traditionally thought to be calcium-dependent phospholipid-binding proteins, is now known to have a more complex set of functions. Annexin interacts with various cell-membrane components that are involved in the structural organization of the cell, intracellular signaling by enzyme modulation and ion fluxes, growth control, and they can act as atypical calcium channels.20 RJ has been said to contain a queen determination factor, so it is speculated the protein plays a role in one of the signaling factors for larvae development. However, we cannot rule out the possibility that the protein may have originated from the disruption of some cells of larvae during the manipulation of this material, which contaminated the samples, resulting in being detected and identified together with the RJ. The lightharvesting complex protein LHCG4 is, as the name implies, a light-harvesting protein, and is the main energy absorber for photosynthesis in green plants.21 The presence of the proteins proves at the molecular level that RJ is a plant-based food source. Alpha-glucosidase was also identified for the first time in RJ by a proteomics approach. The enzyme is secreted from hypopharyngeal gland of the worker bee and functions as hydrolase for alpha glycosidic linkage.22 3.2.2. Comparative Time-Course Protein Profiling and Seasonal Effects by 1-DGE. By comparison of six (three time points for each season) RJ samples, it was determined that there is almost no difference (at the level of silver nitrate or CBB 3224


Figure 3. 1-DGE profile of RJ proteins. Total soluble proteins at 24-72 h after 1st and 2nd collections were separated on 12.5% SDS-PAG as described in section 2.3. The proteins (5 µg/lane) in panel A were stained with silver nitrate (Plus One Silver Staining Kit), and the proteins (40 µg/lane) in panel B were stained with CBB R-250. The molecular weight markers are indicated on the left-hand side of gel 1. The proteins (of 1st and 2nd collections at 48 h; 3 lanes each) stained with CBB R-250 were separated into 14 regions (marked by red boxes and labeled 1-14) and analyzed for their protein components by LC-MS/MS. Two different (from the 1st collection) bands in the 2nd collection (labeled 2-4 and 2-5, respectively; marked in blue boxes) were also excised for MS analysis. Results of the MS identifications are presented in Tables 1 (RJ 1-48) and 2 (RJ 2-48).

staining) in the protein patterns at 24, 48, and 72 h sampling times, on 1-D gels, used in the present study. However, a clear difference in the RJ protein pattern was observed between the first and second collections of RJs (bands 4 (+ 2-4) and 5 (+ 2-5), Figure 3). We identified and compared these proteins by MS, and obtained 8 different protein IDs. Identified proteins from only RJ 1-48 sample were KIAA1904 protein, gametogenetin, ACP54A1, and similar to glucocerebrosidase precursor isoform 2. On the other hand, from the RJ 2-48 sample, we could identify alpha-glucosidase, MRJP 5 precursor, sof2460 precursor, and a protein similar to Y4C6B.6 proteins. As three proteins were found only in the second collection, this may be attributed to the seasonal differences when flowers were different, but we need further evidence to prove this point. However, in general, based on the largely similar protein composition of these two differential (in amounts) bands in the two collections, it can be suggested that the RJ protein composition is stable. Moreover, at both the RJ collection seasons, all the queen honey bee larvae showed normal growth, so these differences are not important for queen determination, but it needs further examination in the future. 3.3. 2-DGE. All the 6 proteins samples from the first and second collections were subjected to 2-DGE, and the silver nitrate-stained gel profiles are presented in Figure 4. Total soluble proteins were separated as described in section 2.4. To minimize spot overlapping, we used 100 µg of protein sample and a narrow range (pH 4-7, 24 cm) IPG strip. To achieve maximum detection of the separated proteins, silver nitrate was used for staining. The 2-D gel profiles show that most of the proteins are in the pH range of 4.5-7 and between approximate molecular masses of 30 and 100 kDa. The ImageMaster software


research articles

Figure 4. Comparison of the 2-DGE profiles of RJ proteins at 24, 48, and 72 h between the 1st and 2nd collections. Top three gels, 1st collection (in red); and bottom three gels, 2nd collection (in blue). Total protein (100 µg) was loaded onto 24 cm, pH 4-7 precast IPG strip followed by SDS-PAGE on a 12-14%T precast ExcelGel XL SDS-PAG. For details see section 2.4. Separated proteins on gel were stained with silver nitrate. The molecular weight markers and pI are indicated on the top left-hand side and top of gel 1, respectively. Downstream image analysis was performed using the ImageMaster 2D Platinum software ver. 5.0. The total number of protein spots detected on the 2-D gel is given at the bottom right-hand corner of each gel. The green arrow indicates the differentially expressed protein spot between the 1st and 2nd collections, respectively.

analysis of gel detected around 348-483 protein spots. Similar to the 1-D gel profile, there was no major difference among the three time points in each collection. However, as observed on the 1-D gel, in the second collection, a spot (basic region, marked by green arrow) of around 60 kDa showed increase in amount as compared to the first collection 2-D gel profile. 3.3.1. The 2-D Gel Reference Map. The 48 h RJ first collection sample was also used as a representative for creating the 2-D gel reference map (Figure 5). We excised only the major and highly reproducible 147 protein spots. The gel spots were excised for in-gel digestion with trypsin. The tryptic peptides were analyzed by nESI-LC-MS/MS and the proteins identified using MASCOT search engine and Swiss-Prot/NCBI nonredundant protein databases are listed in Table 3. A total of 105 (6 in 105 were identified with proQ DPGS and MS analysis, see section 3.4) proteins were identified representing 14 (1 in 14 were identified by proQ DPGS and MS analysis, see section 3.4) nonredundant proteins. Proteins are color-coded as red, MRJP 1 (49 protein spots; 46%); orange, MRJP 2 (14 protein spots; 13%); yellow, MRJP 3 (12 protein spots; 11%); blue, MRJP 4 (5 protein spots; 5%); green, MRJP 5 (4 protein spots; 4%); pink, glucose oxidase (4 protein spots; 4%); sky blue, others. Protein spot numbers are the same as mentioned in Table 3. The identified proteins by 2-DGE in combination with MS analysis were categorized on a pie chart presented in Figure 6. The pie chart reveals that 86% of the identified proteins belonged to the MRJP family. MRJPs were visualized at many different points on the 2-D gel. Previously, it was reported that MRJPs are produced in the hypoharyngeal gland and secreted into the RJ, where some of these proteins may suffer glycosylation and/or partial proteolysis, generating new isoforms of

these proteins in the stored RJ.15 This point is validated on the 2-D gel presented in this study. 3.3.2. The Novel Proteins Identified by 2-DGE. Other than the MRJP family proteins mentioned in section 3.2.1.1, and previous papers, we identified some novel proteins on 2-D gel, which are discussed below. Spot no. 102 was identified as vicilin, which has been described as a seed storage protein and a major lentil allergen.23 In addition, vicilin mRNA was detected by RT-PCR in the pollen of Pisum sativum.24 Recently, surfaceexposed IgE-binding epitopes of close overall conformation were characterized on the molecular surface of three-dimensional models built for the vicilin allergens of peanut (Ara h 1), walnut (Jug r 2), hazelnut (Cor a 11), and cashew nut (Ana o 1), and it was shown that vicilin has a glyco-epitope.25 Our finding suggests that RJ (sample used in the present study) includes a vicilin-like protein, which is derived from flower pollens collected by worker honey bees. This is a potentially important finding as it indicates that RJ may contain proteins as possible allergens. This is also the first report of the identification of an allergic proteinaceous factor in RJ. Spot no. 145 was identified as an odorant binding protein (OBP) 14. OBPs are soluble proteins interacting with odor molecules and pheromones in the perireceptor areas, the sensillar lymph in insects.26 The remarkable olfactory power of insect species is thought to be generated by a combinatorial action of two large protein families, G protein-coupled olfactory receptors (ORs) and OBPs. In olfactory sensilla, OBPs deliver hydrophobic airborne molecules to ORs, but their expression in nonolfactory tissues suggests that they also may function as general carriers in other developmental and physiological processes, a monophyletic subfamily of OBPs where the diversification of some Journal of Proteome Research • Vol. 7, No. 8, 2008 3225

research articles

Furusawa et al.

Figure 5. Development of a high-resolution 2-D gel reference map of RJ proteins. The 48 h RJ 1st collection sample was used as a representative for creating the 2-D gel reference map. Total soluble proteins were separated on IPG strips (24 cm, pH 4-7) in the first dimension followed by (12-14%) ExcelGel SDS-PAGE in the second dimension as described in Figure 4. Proteins were visualized with silver nitrate. A total of 105 protein spots were excised and in-gel digested. The tryptic peptides were analyzed on the nano electrospray ionization mass spectrometer (nESI-LC-MS/MS) and proteins were identified using MASCOT search engine and Swiss-Prot/NCBI nonredundant protein databases. Proteins are color-coded as red, Major Royal Jelly protein (MRJP) 1; orange, MRJP 2; yellow, MRJP 3; blue, MRJP 4; green, MRJP 5; pink, Glucose Oxidase; sky blue, others (see Figure 6; protein spot numbers are the same as mentioned in Table 3).

Figure 6. Pie diagram indicating the major RJ protein (MRJP) family as the dominant proteins in RJ on 2-D gel. A total of 105 (6 in 105 were identified with proQ DPGS and MS analysis, see also Figure 7B) proteins were identified from 2-D gel (12-14% gradient and stained with silver nitrate) as described in Figure 5. These consisted of 84 proteins (86%) belonging to MRJP family (in black), four novel proteins (7%, in green), and four predicted proteins (7%, in blue).

amino acids appears to have been accelerated by positive selection.27 It is tempting to speculate a role for this protein as a sensor in larvae for amino acid reception; however, it may be just another protein from the honey bee in RJ. Spot no. 140 was identified as albumin. The physicochemical characteristic of the RJ protein resembles albumin. The essential amino acids rate of the RJ proteins is equal to other nutritional proteins (milk casein 49.1%, chicken egg white albumin 51.6%).18 This study also identified some predicted proteins based on their homology. Spot nos. 29 and 30 were identified as glucocerebrosidase precursor isoform 2 and spot no. 57 was identified as glucose dehydrogenase isoform 1. Glucocerebrosidase (EC 3.2.1.45) is a membrane-bound lysosomal enzyme which catalyzes the hydrolysis of glucocerebroside.28 These enzymes may be derived from the worker honey bee, and might 3226


help digest RJ components for the developing larvae. Spot nos. 121 and 143 were identified as similar to ferritin 1 heavy chain homologue (HCH) CG2216-PE, isoform E, and spot no. 138 was identified as similar to ferritin 2 light chain homologue (LCH) CG1469-PA, isoform A. Insect ferritins are composed of two types of subunits, homologues of the vertebrate H and L subunits. These are designated as heavy and light chain homologues (HCH and LCH), respectively.29,30 Iron transport is important for larvae development, and ferritin can accommodate thousands of iron atoms in the form of a ferrihydrite mineral. This high capacity for iron storage underlies the central role these proteins play in iron metabolism of plants and animals. 3.4. Potential PTMs in the RJ Proteome. In this study, we detected the phosphoprotein and glycoprotein modifications


research articles

Figure 7. Potential post-translational modifications in the RJ proteome. Pro-Q diamond phosphoprotein and glycoprotein gel stain (Pro-Q DPGS/Pro-Q EGGS) was used to detect the RJ phosphoproteins/glycoproteins on 1- and 2-D gels. Proteins from the 1st collection (1-24, -48, -72 h; 40 µg each lane) were separated on 12.5% SDS-PAG (A), and the 48 h RJ sample was further resolved on 18 cm (pH 4-7) IPG strips in the first dimension and 12.5% hand-cast gel in the second dimension (B). In A, phosphoproteins (single band of ca. 50 kDa) and glycoproteins (between 42 and 97 kDa, and above 180 kDa) were stained with Pro-Q DPGS (left) and Pro-Q EGGS (center), followed by SYPRO Ruby (right) gel stain for staining total protein. In the gel boxed in pink, the arrow in pink shows the phosphoprotein markers and phosphoproteins, and the arrows in green on the right-hand side indicate the glycoprotein markers and glycoproteins, respectively. In B, 6 arrows (in pink) indicate the potential phosphoproteins on 2-D gel (upper), which were excised under UV light and poststained with SYPRO Ruby (bottom) to reveal their position just under the MRJP1 family proteins (see also Figure 5). MS analysis of these phosphoprotein spots revealed their identity as MRJP 7 (p1 to p4) and MRJP 2 (p5 and p6), respectively.

in RJ protein extracts using ProQ DPGS and ProQ EGGS stains, respectively (Figure 7). The staining intensities on the 1-D gel suggested that most of the RJ proteins are probably glycoproteins (Figure 7B), one band was specifically stained with ProQ DPGS (Figure 7A). We can speculate on the nature of these glycoproetins based on our 1-/2-DGE MS analysis data and previous reports. Many of the visualized glycoprotein bands are in molecular weight range of 50-100 kDa. Two glycoproteins in RJ, namely, a 350 kDa protein named apisin, and 57 kDa protein were previously reported.31,32 Apisin consists of six subunits (58 000 Da), and the carbohydrate chain is a high mannose type. The protein shows cell proliferation activity and suppression cell death.31 The 57 kDa protein also shows same activity, though comparatively weaker.32 This may indicate that many of the visualized glycoproteins were apisin fragments or the 57 kDa protein. From 1-DGE data, visualized and stained

glycoproteins were at same position as the MRJP family, so it can be suggested that the MRJP family proteins are glycosylated. Using a homogeneous 12.5% 2-D gel in the second dimension, we could separate the potential phosphoproteins as two separate regions on the gel. One region was found to be present just behind the abundant MRJP1 (spots 1-8) and the second was similar to the spots 9 and 11 (MRJP2), on the gradient 2-D gel. The protein was excised under UV light and MS analysis identified these phosphoproteins as two MRJP family members, namely, MRJP2 and MRJP7. MRJP 2 carries potential phosphorylation sites with high score: 5 (Ser), 4 (Thr), and 6 (Tyr) score (NetPhos 2.0), further suggesting that the protein may be phosphorylated. MRJP 7 also carries potential phosphorylation sites with high score: 11 (Ser), 9 (Thr), and 9 (Tyr) high score (NetPhos 2.0). If in future studies, we can identify the phosphorylation sites in MRJP2 and 7; this will be a first such report Journal of Proteome Research • Vol. 7, No. 8, 2008 3227

research articles for the MRJP family. The functions of MRJP 2 and 7 are not known; however, these proteins could be speculated as mineral binding protein such as casein, and promote absorption of minerals for the queen honey bee larvae. These identifications need to be physiologically confirmed in order to clearly understand the functional role of these proteins in queen honeybee development and/or promote human health and beauty.

4. Conclusions and Future Perspectives RJ is necessary for the developing honey bee queen, and is now widely used for human health and beauty. We fulfilled the first goal of the study, by identifying the largest number of RJ proteins to date, including many novel proteins and the PTMs, by a combination of 1- and 2-DGE in conjunction with MS. In this study, by comparison of six RJ samples at two different collections, we determined that the expression of RJ proteins did not change with the collection period used commercially, namely, at 24 to 72 h. However, we found that among the two closely occurring collections seasons (first and second collection, respectively) there were two major differences in proteins bands/spots between these RJs. Though the different protein composition may be attributed to the different flower blooms, we cannot precisely impress upon the readers as to why this difference exists. However, if we can find such difference among numerous samples, then we might be looking at a potential marker for RJ. Can it be used for the evaluation of the RJ? We will only know this after further detailed studies. We would like to mention another aspect of the proteomics approach untested in the present study. As we have identified the MRJP family as the major abundant proteins in the RJ, it is reasonable to look for the low-abundance proteins in the RJ. In the future, with an aim to build a more detailed RJ Proteomics Database, we hope to use MudPIT (Multidimensional Protein Identification Technology) and gel filtration techniques to enrich these presently unknown low-abundance proteins in the RJ. We name it as the “Hidden RJ Proteome”, and in the future, when we identify these proteins, it will help us gain insight into the true functions of the RJ with respect to its role in the development of the queen honey bee. 4.1. RJ Potentiality. From the proteomics perspective, our results suggest that RJ includes potential signals for determination of the queen honey bee. So far, there are hypothesis as to why the larva which continued to eat RJ will become the queen honey bee.33 One viewpoint is the “determination concept”.33 It was thought that The Rj contained a special factor which decided the fate of larvae to develop into a queen bee; however, this desirable factor has not been identified. The other viewpoint is the “balanced-composition concept”, where the total components of RJ are connected for differentiation to the queen.33 From the various proteins that were identified by our study, it can be reasoned that the RJ prepares the larvae to become a queen by physical and biological stimulation with its components rather than one special factor. Simth and coworkers reported that the RJ proteins are divided into 4 components, namely, (i) enzymes, (ii) nutrition proteins, (iii) defense-related proteins and peptides, and (iv) physiology functional proteins and peptides.18 In addition, we could identify the proteins, such as vicilin and Light-Harvesting complex Protein LHCG4, which may be indicative of RJ collection area/season, and characteristic of the bee colony; we propose these as the “fifth component” as (v) colony-marker proteins. On the other hand, for humans, these results suggest 3228


Furusawa et al. potential of not only danger via allergic reactions, but also the health/beauty promoting function of RJ. It is important evidence, which supports the fact that the RJ has not been certified as Substances Generally Recognized As Safe (GRAS) by the Food and Drug Administration (FDA), and as a functional food in Japan.34 It is necessary for RJ researchers to know the various functional aspects (read protein complement) on RJ, and propose an appropriate usage of RJ. We hope the RJ proteome data will be used by researchers in food functional studies and the companies that market RJ, ultimately hoping that the RJ research will be fruitful for human life and insect biology.

Acknowledgment. The close cooperation and support during RJ collection from the farm staff and the Kato Bihoen Honpo Co., Ltd is greatly appreciated. The work was partly supported by a grant from the Japan Royal Jelly Fair Trade Council. This work was also partly supported by a grant from the Korea Science and Engineering Foundation through Protein Network Research Center at Yonsei University (Grant No. R112000078010010). R.R. and G.K.A. highly appreciate the vision of Prof. V. P. Agrawal (Founding Director, RLABB, Nepal) in establishing collaboration with Prof. Yu-Sam Kim at Yonsei University, Korea. T.F. greatly appreciates the kind support and encouragement from Dr. Hitoshi Iwahashi (HTRC). Supporting Information Available: Figures showing the polymorphism of the MRJP 3 protein and the high similarity of MRJP family components. Tables listing the Pro-Q diamond staining for phosphoproteins and the Pro-Q emerald 300 staining for glycoproteins. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Honeybee Genome Sequencing Consortium Nature 2006, 26, 931949. (2) Roberts, S. P.; Elekonich, M. M. J. Exp. Biol. 2005, 208, 4193–4198. (3) Winston, M. L. The Biology of the Honeybee. Harvard University Press: Cambridge, MA, 1987. (4) Robinson, G. E. In Neurobiology and Behavior of Honeybees; Menzel, R., Mercer, R. , Eds.; Springer-Verlag: New York, 1987; pp 266-279. (5) Fujii, A. Honeybee Sci. 1995, 16, 97–104. (6) Agrawal, G. K.; Yonekura, M.; Iwahashi, Y.; Iwahashi, H.; Rakwal, R. J. Chromatogr., B 2005, 815, 109–123. (7) Agrawal, G. K.; Yonekura, M.; Iwahashi, Y.; Iwahashi, H.; Rakwal, R. J. Chromatogr., B 2005, 815, 125–136. (8) Agrawal, G. K.; Yonekura, M.; Iwahashi, Y.; Iwahashi, H.; Rakwal, R. J. Chromatogr., B 2005, 815, 137–145. (9) Scarselli, R.; Donadio, E.; Giuffrida, M. G.; Fortunato, D.; Conti, A.; Balestreri, E.; Felicioli, R.; Pinzauti, M.; Sabatini, A. G.; Felicioli, A. Proteomics 2005, 5, 769–776. (10) Scho¨nleben, S.; Sickmann, A.; Mueller, M. J.; Reinders, J. Anal. Bioanal. Chem. 2007, 389, 1087–1093. (11) Li, J.; Wang, T.; Zhang, Z.; Pan, Y. J. Agric. Food Chem. 2007, 55, 8411–8422. (12) Agrawal, G. K.; Rakwal, R. Mass Spectrom. Rev. 2006, 25, 1–53. (13) Furusawa, T.; Rakwal, R.; Nam, H. W.; Hirano, M.; Shibato, S.; Kim, Y. S.; Ogawa, Y.; Yoshida, Y.; Kramer, K. J.; Kouzuma, Y.; Agrawal, G. K.; Yonekura, M. J. Proteome Res. 2008, 7, 938–959. (14) Agrawal, G. K.; Thelen, J. J. Proteomics 2005, 5, 4684–4688. (15) Santos, K. S.; dos Santos, L. D.; Mendes, M. A.; de Souza, B. M.; Malaspina, O.; Palma, M. S. Insect Biochem. Mol. Biol. 2005, 35, 85–91. (16) Drapeau, M. D.; Albert, S.; Kucharski, R.; Prusko, C.; Maleszka, R. Genome Res. 2006, 16, 1385–1394. (17) Kucharski, R.; Maleszka, R.; Hayward, D. C.; Ball, E. E. Naturwissenschaften 1998, 85, 343–346. (18) Simu ´ th, J.; Bı´likova´, K. Honeybee Sci. 2004, 25, 53–62. (19) Peiren, N.; Vanrobaeys, F.; de Graaf, D. C.; Devreese, B.; Van Beeumen, J.; Jacobs, F. J. Biochim. Biophys. Acta 2005, 31, 1–5. (20) Moss, S. E.; Morgan, R. O. GenomeBiology 2004, 5, 219.

research articles

RJ Proteomics Reveals Novel RJ Proteins and Phospho/Glycoproteins (21) Consoli, E.; Croce, R.; Dunlap, D. D.; Finzi, L. EMBO Rep. 2005, 6, 782–786. (22) Ohashi, K.; Sawata, M.; Takeuchi, H.; Natori, S.; Kubo, T. Biochem. Biophys. Res. Commun. 1996, 16, 380–385. (23) Sanchez-Monge, R.; Lopez-Torrejo´n, G.; Pascual, C. Y.; Varela, J.; Martin-Esteban, M.; Salcedo, G. Clin. Exp. Allergy 2004, 34, 1747– 1753. (24) Zakharov, A.; Giersberg, M.; Hosein, F.; Melzer, M.; Mu ¨ ntz, K.; Saalbach, I. J. Exp. Bot. 2004, 55, 1463–1471. (25) Barre, A.; Sordet, C.; Culerrier, R.; Rance´, F.; Didier, A.; Rouge´, P. Mol. Immunol. 2008, 45, 1231–1240. (26) Pelosi, P. Ann. N.Y. Acad. Sci. 1998, 855, 281–293. (27) Foreˆt, S.; Maleszka, R. Genome Res. 2006, 16, 1404–1413. (28) Tsuji, S.; Choudary, P. V.; Martin, B. M.; Winfield, S.; Barranger, J. A.; Ginns, E. I. J. Biol. Chem. 1986, 5, 50–53.

(29) Dunkov, B.; Georgieva, T. Insect Biochem. Mol. Biol. 2006, 36, 300– 309. (30) Nichol, H.; Law, J. H.; Winzerling, J. J. Annu. Rev. Entomol. 2002, 47, 535–559. (31) Kimura, M.; Kimura, Y.; Tsumura, K.; Okihara, K.; Sugimoto, H.; Yamada, H.; Yonekura, M. Biosci. Biotechnol. Biochem. 2003, 67, 2055–2058. (32) Kamakura, M.; Fukuda, T.; Fukushima, M.; Yonekura, M. Biosci. Biotechnol. Biochem. 2001, 65, 277–284. (33) Matsuka, M. Honeybee Sci. 1998, 19, 1–8. (34) Swinbanks, D.; O’Brein, J. Nature 1993, 364, 180.

PR800061J


Comprehensive Royal Jelly (RJ) Proteomics Using ... - ACS Publications

Recommend Documents