Project PXD010898

PRIDE Assigned Tags:
Biological Dataset

Summary

Title

Honeybee hypopharyngeal glands LC-MS/MS

Description

Royal jelly (RJ) is a proteinaceous secretion of the hypopharyngeal glands (HGs) in the head of honeybee workers. It is a critical food for queen bees and young larvae that decides the fate of fertilized eggs in developing into either queen bees or worker bees during the early larval stages. RJ is also widely used in humans for health promotion as agent, such as antibacterial, antioxidant, and antiaging properties. To increase RJ yields, a stock of high RJ producing honeybees (RJBs) has genetically selected from Italian honeybees (ITBs) in China since 1980s. To date, one colony of RJBs can produce more than 10 kg of RJ per year, a yield that is 10-times greater than that produced by a colony of ITBs. To elucidate the mechanism of the enforced gland performance in producing RJ in RJBs, the spatio-temporal HG proteomes of newly emerged bees, nurse bees, and forager bees, were compared between the ITBs and RJBs. Proteins in the critical pathways that are implicated in the secretory activity of RJ in HGs are validated biochemically and biologically by manipulating the NBs into extended nursing periods and the FBs to revert into NBs. This will provide a novel mechanistic insight into the HGs achieving an enhanced biological mission of producing the valuable bee-product, RJ.

Sample Processing Protocol

Protein extraction and digestion In short, prior to protein extraction the hypopharyngeal gland tissue was homogenized in liquid hydrogen with a pestle. The HGs were then mixed with a lysis buffer containing 8 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 20 mM Trisbase, 30 mM dithiothreitol (DTT), 2% Bio-lyte (pH 3–10), and protease inhibitors (Roche-04693116001,Basel, Switzerland) in ice for 30 min. Afterwards, the sample was further centrifuged at 15,000 g for 20 min at 10 °C to remove the insoluble fractions. Three volumes of ice-cold acetone were added to the recovered supernatant at -20 °C for 4 h to precipitate the proteins. Subsequently, the protein pellets were centrifuged at 8,000 g at 10 °C for 20 min. The supernatant was discarded, followed by extraction of the protein pellet at room temperature (RT). The recovered proteins were re-suspended in 100-150 μL of 5 M urea, and protein concentration was quantified by Bradford assay. Of each sample, 200 μg protein was used by adding four volumes of 40 mM NH4HCO3, mixed with DTT (final concentration 10 mM) for 1h, and then alkylated with iodoacetamide (final concentration 50 mM) for 1h in the dark. To digest protein into peptides, sequencing grade modified trypsin (modified sequencing grade, Cat. # V7491, Promega, Medison, WI, USA) was used (enzyme/protein ratio of 1:100 (W/W)) at 37°C for 14 h. The enzymatic digestion was stopped by adding 1 μL of formic acid to the solution. The digested peptide samples were desalted by C18 column (Agilent Technologies Inc., USA). The eluted peptide solution was collected and extracted using a SpeedVac system (RVC 2-18, Marin Christ, Osterod, Germany) and stored at -80 °C for subsequent LC-MS/MS analysis. LC−MS/MS analysis The digested peptide samples were re-dissolved in 50 µL of 0.1% FA. Three replicates of each sample were run using Q-Exactive mass spectrometer (Thermo Fisher Scientific) and coupled to the EASY-nLC 1000 system using a nano electrospray ion source (Thermo Fisher Scientific, USA). To enrich the peptide samples, they were first loaded onto a 2 cm long trap column (75 μm inner diameter fused silica containing 3 μm Aqua C18 beads, Thermo Fisher Scientific, USA) for 2 min in buffer A (0.1% acetic acid) at a flow rate of 10 μL/min. Secondly, the peptides were separated by analytical column (15 cm long, 50 μm inner diameter fused silica column filing with 2 μm Aqua C18 beads, Thermo Fisher Scientific, USA) using a 120 min gradient. Peptides were gradient eluted in 110 min with a linear gradient from 8% to 30% acetonitrile at a flow rate of 300 nL/min. The eluting peptides from the analytical column were directly infused into a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA) via electrospray ionization. The settings for a data-dependent mode to collect the MS and MS/MS data were as follows: one full scan (resolution 70,000 at 400 m/z; 350 to 1600 m/z) followed by top 20 MS/MS scans using higher-energy collisional dissociation in the linear ion trap mass spectrometer (resolution: 15,000, isolation window: 2 m/z, normalized collision energy: 28) using dynamic exclusion (charge exclusion: unassigned 1, >8; peptide match: preferred; exclude isotopes: on; dynamic exclusion: 30s).

Data Processing Protocol

Identification and abundance level quantitation of proteins The MS/MS data in RAW were retrieved using Xcalibur (version 3.0, Thermo Fisher Scientific, USA) and searched using in-house PEAKS software (version 8.5, Bioinformatics Solutions Inc., CA). A database containing protein sequences of Apis mellifera, including common contaminants was downloaded from NCBI and used, totaling to 22,757 entries. The parameters of the search database was as follows: trypsin; maximum missed cleavage: 2; fixed modification: precursor ion and MS/MS tolerances: 15 ppm and 0.05 Da; enzyme specificity: carbamidomethyl (C, +57.02); and variable modification: oxidation (M, +15.99). The fusion-decoy database search strategy with threshold FDR ≤ 1% was used to control the false discovery rate (FDR) at both the protein and peptide levels (45). A protein was considered as identified only if it had at least two unique peptides. To quantify the relative level of protein abundance of HGs in both ITBs and RJBs, three replications of each sample were applied in the quantification module of PEAKS software (version 8.5) via label-free strategy. Feature detection was performed separately on each sample using the expectation-maximization algorithm. Using the high-performance retention time alignment algorithms, the features of the same peptide from three replicates of each sample were reliably aligned. Normalization was done by dividing each matrix by a factor of the samples obtained as follows: the total ion current (TIC) of the individual sample/ the TIC of the reference sample. Quantification of the protein abundance levels of HGs in all samples of both bee lines was done using the sum of the three highest ion peak intensities of the tryptic peptides. Furthermore, the protein abundance levels were compared over NEs, NBs and FBs in each bee stock and between each time point of NEs, NBs and FBs of both bee lines. GO term enrichment analysis To understand the biological implications of the identified proteins in the HGs of the worker honeybees, the identifiers of protein GI numbers were used as an input for GO term enrichment (functional classes and pathway) using ClueGOv2.3.2, a Cytoscape plug-in (http://www.ici.upmc.fr/cluego/). The number of proteins identified from the samples was compared with the number of functionally GO annotated proteins in the entire honeybee (Apis mellifera. L) genome for enrichment analysis. A right-sided hyper-geometric test was used to report the significantly enriched functional GO categories and pathways in biological processes. FDR was calculated using Bonferroni step-down test to correct the P-value. The GO terms and pathways were considered as significantly enriched only when the corrected P-value was < 0.05. The kappa score (0.4) in ClueGo was mandatory to link the nodes in the functional networks.

Contact

Han Hu, Chinese Academy of Agricultural Sciences, Institute of Apicultural Research
Jianke Li, Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, No. 1 Beigou Xiangshan, Beijing, 100193, China ( lab head )

Submission Date

28/08/2018

Publication Date

11/01/2019

Instrument

Q Exactive

Software

Not available

Quantification

Not available

Experiment Type

Shotgun proteomics

Publication

    Hu H, Bezabih G, Feng M, Wei Q, Zhang X, Wu F, Meng L, Fang Y, Han B, Ma C, Li J. In-depth Proteome of the Hypopharyngeal Glands of Honeybee Workers Reveals Highly Activated Protein and Energy Metabolism in Priming the Secretion of Royal Jelly. Mol Cell Proteomics. 2019 PubMed: 30617159