PXD000592

PXD000592 is an original dataset announced via ProteomeXchange.

Title	Mouse Core Acrosomal Matrix LC-MSMS
Description	Some molecular and cellular mechanisms of fertilization are still not completely described. The role of the acrosomal matrix (AM), the particulate compartment within the acrosome, is one of them. Here, we proposed that amyloids, highly ordered protein aggregates, by their physical and chemical properties could contribute to AM roles in fertilization. Purified AM were exposed to a two-step extraction to sequentially strip off soluble proteins. The remaining insoluble material (core) was subjected to a mass spectrometry analysis. Preparation of samples for MS analysis: Three different approaches were used to optimize identification of peptides in the AM core. For in-gel digestion, core samples were resuspended in 13.2mM SA pH3 containing 8M urea and 100 mM DTT and incubated for 1 hr at RT. Proteins were separated on a hand casted 12% polyacrylamide Tris-glycine gel. After silver staining, visible bands were cut from the gel, destained and washed with ddH2O (2 X 5 min) then with 50% ethanol (2 X 5 min) before stored at -20 degrees C. Gel pieces were tryptically digested in 25mM triethylammonium bicarbonate buffer at 37 degrees C overnight with trypsin. For in-solution digestion, core samples were resuspended in 13.2mM SA pH3/ 8M urea/ 100mM DTT and incubated for 1 h at RT followed by 15 min at 70 degrees C. Iodoacetamide was added to 10mM and proteins were alkylated by incubation for 15 min at RT in the dark. Samples were added to a pre-rinsed spin filter and centrifuged at 14,000 X g. Samples were washed with 9M urea then with 25mM ammonium bicarbonate. Samples were digested with trypsin in 25mM ammonium bicarbonate overnight. After digestion, samples were spin at 14,000 X g and washed 2 times with 25mM ammonium bicarbonate. The retentate was transferred to a new tube and air dried. For on-membrane digestion, the samples were dotted on nitrocellulose membrane and digested by trypsin. Briefly, core samples were resuspended and treated as for in-solution digestion. Then, samples were dotted by gravity on the membrane. Wells were rinsed with 20mM SA pH3 followed by TBS. Dots were cut and air-dried. After the protein digestion with trypsin in 25mM ammonium bicarbonate the membranes were dissolved with acetone and the precipitated peptides were air-dried. All digested peptides were reconstituted in 2% acetonitrile/ 0.1% formic acid for MS analysis. MS data acquisition: Protein identification by liquid chromatography tandem mass spectrometry (LCMS/MS) analysis of peptides was performed using an LTQ Orbitrap Velos MS interfaced with a 2D nanoLC system. Protein and Peptide Identification: MS/MS spectra were extracted using the ProteoWizard Toolkit. The spectra were analyzed using the GPM Manager with X!Tandem to search against a home made mouse database containing 213,054 nonredundant protein sequences created by using mouse sequences from Ensembl database (files Mus_musculus.GRCm38.73.pep.all & Mus_musculus.GRCm38.73.pep.abinitio) and from NCBI database (file nr downloaded on 09/19/2013) . Search was performed using fully tryptic enzyme specificity (See deposited MS data for details). Peptides and proteins that have an expectation value of log10 (e) <= -2 were included in the results. Curated results were obtained by keeping only proteins with at least one mouse-specific matching peptide (peptide match = 100% identities and 100% coverage on a unique mouse protein and <100% identities and/or <100% coverage on human proteins). In addition, trypsin-like proteins were kept if at least one peptide was not matching on exogenous pig trypsins.
HostingRepository	PRIDE
AnnounceDate	2014-05-07
AnnouncementXML	Submission_2014-05-07_07:55:30.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Dr Gail A Cornwall
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	monohydroxylated residue
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2013-11-22 02:17:34	ID requested
⏵ 1	2014-05-07 07:55:31	announced

Guyonnet B, Egge N, Cornwall GA, Functional amyloids in the mouse sperm acrosome. Mol Cell Biol, 34(14):2624-34(2014) [pubmed]

submitter keyword: Mouse, Spermatozoa, Acrosome, Acrosomal matrix, SDS-resistant, amyloid, Proteome

Dr Gail A Cornwall
contact affiliation	Texas Tech University Health Sciences Center, Cell Biology & Biochemistry Department
contact email	benoit.guyonnet@live.fr
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2014/05/PXD000592

PRIDE project URI

• PRIDE
  1. PXD000592
     1. Label: PRIDE project
     2. Name: Mouse Core Acrosomal Matrix LC-MSMS