Dataset Information

Analysis of the honeybee sperm proteome using MudPit and 2D PAGE

ABSTRACT: Honeybee semen was collected by gently squeezing the male abdomens. Samples were pooled and then centrifuged. Pelleted sperm was collected and analysed with both 2D PAGE and gel-free methods. For the 2D PAGE, protein spots were digested using trypsin. Extracted peptides, resolved on a C18 column, were analysed by an Agilent LC/MSD Trap XCT Ultra 6330 mass spectrometer. Spectra were searched against the honeybee protein sequences (RefSeq release 48) using Mascot algorithm (with 1 missed cleavage, Cys-carbamidomethylation as fixed modification, and Met-oxidation and N/Q-deamidation as variable modifications). For the MudPit analysis the sperm sample was digested by trypsin. Peptides were resolved using strong cation exchange chromatography followed by reverse phase (C18) HPLC and finally analysed by an Agilent QTOF mass spectrometer. Resulting spectrum files were converted to mzXML and merged using ProteoWizard msconvert. These were then searched against honeybee protein sequences using Mascot, Omssa and X!tandem (all with 1 missed cleavage, and Met-oxidation and N/Q-deamidation as variable modifications). Results of the three search engines were pooled using TPP.

INSTRUMENT(S): 6510 Quadrupole Time-of-Flight LC/MS, 6330 Ion Trap LC/MS

ORGANISM(S): Apis Mellifera (honeybee)

TISSUE(S): Spermatozoon

SUBMITTER: Reza Zareie

LAB HEAD: Boris Baer

PROVIDER: PXD000169 | Pride | 2020-01-17

REPOSITORIES: Pride

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Publications

Long-term survival of high quality sperm: insights into the sperm proteome of the honeybee Apis mellifera.

Zareie Reza R Eubel Holger H Millar A Harvey AH Baer Boris B

Journal of proteome research 20131015 11

In the social bees, ants, and wasps, females (queens) mate only during a brief period early in their lives and afterward store a lifetime supply of sperm in a specialized organ, the spermatheca. In some species, stored sperm can remain viable for several decades and is used by queens to fertilize millions of eggs. The physiological adaptations that allow this prolonged survival are unknown. To unravel them, we conducted proteomic analyses on the sperm of the honeybee Apis mellifera to define pro ...[more]

PMID: 24127784

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Project description:The honeybee brain is comprised of a nervous system that sufficiently regulates this life transition. Knowledge about how protein phosphorylation functions in regards to the neurobiological activities in the honeybee brain to drive the age-specific labor division is still lacking. Protein phosphorylation, the most common post-translational modification (PTM), is a key switch for rapid on-off control of signaling cascades that regulate cell differentiation and development, enzyme activity and metabolic maintenance in living cells. A fundamental mechanism for regulating signaling network and protein activity is the covalent PTM of serine (Ser), threonine (Thr), and tyrosine (Tyr) residues with phosphate. Fortunately, because of advances in phosphopeptide enrichment and improvements in mass spectrometry (MS) instrumentation and methods, phosphoproteomics has enabled large-scale identification of protein phosphorylation sites and phosphorylation networks in biological samples. Although the proteome has been mapped in the brain of nurse and forager bees, knowledge about age-specific effects of phosphorylation regulation on proteins in the honeybee brain is still lacking. Moreover, information in regards to the honeybee phosphoproteome is also very limited. Only very recently, in-depth phosphoproteomics analyses of protein phosphorylation networks in the hypopharyngealgland of the honeybee have been reported. Although the phosphoproteome analyses during the development of brood and salivary glands has been reported, only very limited proteins were phosphorylated and phosphorylation sites of those phosphoproteins were not assigned. Therefore, a comprehensive characterization of phosphoproteomics and changes in the brains of nurse and forager bees is key to understand the phosphorylation events underlying age-specific physiology to achieve the completion of biological missions in this well-organized social community of the honeybee. Honeybee (A. m. ligustica) colonies used for sampling were raised at the apiary of the Institute of Apicultural Research, Chinese Academy of Agricultural Science, Beijing. Newly emerged (<12 h after emergence) worker bees were marked on their thoraxes and placed back into the colonies to develop and then the marked nurse and forager bees were collected on days 10 and 20, respectively. There were 150 bees sampled from each of the five colonies which have queens at the same age. In brief, for each time point, worker bees were sampled from five colonies, and pooled all samples for further analysis. This procedure was repeated three times, so that we finally ended up with three independent biological replicates per time point, each consisting of 150 honeybees. Then their brains were dissected, and the brain samples were pooled and stored at −80 °C for further analysis. All the colonies were managed with almost identical population, food, and brood during the nectar flow of chaste berry (Vitexnegundo L.) in June.

Dataset Information

Analysis of the honeybee sperm proteome using MudPit and 2D PAGE

Publications

Long-term survival of high quality sperm: insights into the sperm proteome of the honeybee Apis mellifera.

Similar Datasets

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