PXD048334

PXD048334 is an original dataset announced via ProteomeXchange.

Title	Quantitative analysis of ribosomal protein expression in bacteria
Description	Analysis of ribosomal proteins purified as ribosome particles. Two set of samples were analyzed: 1) ribosomes purified from bacteria expressing sgRNA that does not target any ribosomal RNA gene (control); 2) ribosomes purified from bacteria expressing sgRNA targeting three ribosomal RNA genes. The aim of the project is to quantify the peptides and compare both sets of samples. The ribosomal peptides prepared from the bacteria expressing the sgRNA targeting the rRNA genes are expected to be less abundant than in the control samples. The two sample sets are the following: A-D (four biological replicates) are the samples from the bacteria expressing functional sgRNA downregulating three rRNA genes. E-H (four biologicial replicates) are the control samples from the bacteria expressing sgRNA that do not target any rRNA gene. All the sampes were spiked with an almost equivalent amount of ribosomes from a WT strain grown in almost 100% 13C and 15N labelled minimum medium. Ribosomes were purified according to published protocols. Basically, the ribosome particles are sedimented by ultra-centrifugation in a saccharose-rich medium. The recovered pellets were resuspended for electrophoretic migration (stacking gel only) for later in-gel trypsinolysis. The obtained peptides were extracted from the gel according to standard procedures and analyzed by LC-MS/MS on a QExactivePlus Orbitrap mass spectrometer (Thermo scientific). The raw data were converted to mzXML (raw data files) and processed using our proteomics software suite (i2MassChroQ, see http://pappso.inrae.fr/en/bioinfo/). The database search engine was X!Tandem, that produces, for each MS run file analyzed, a corresponding XML file, that we submit here as the database search identification data files). The X!Tandem program was first configured to run without accounting for labelled peptides (one set of "light-only" XML files were thus generated by X!Tandem). Then, X!Tandem was configured to account for all the residues labelled 100% with 13C and 15N (one set of "heavy-only" XML files were thus generated by X!Tandem). We loaded first the "light-only" X!Tandem-generated XML files in i2MassChroq, checked the quality of the data and wrote to disk an XPIP (i2MassChroq project file) file for the four replicates of both sample sets all taken together. Then we did this likewise for the "heavy-only" X!Tandem-generated XML files (at this time we configured i2MassChroQ to look for heavy-isotope-labelled peptides). We finally open the two XPIP files (light-only and heavy-only) and save the combination of the data to a new XPIP file (light-and-heavy). The quantitative analysis was performed inside the i2MassChroq's MassChroQ module which is then directed to write a spreadsheet file with all the identification/quantification data of the peptides. That spreadsheet file was then used within GNU-R to perform data reformatting and peptide quantification work (comparison of the amount of selected peptides belonging to known ribosome subunits between the two data sets).
HostingRepository	MassIVE
AnnounceDate	2024-01-12
AnnouncementXML	Submission_2024-01-12_14:27:14.657.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	rusconi
SpeciesList	scientific name: Escherichia coli; common name: E. coli; NCBI TaxID: 562;
ModificationList	CarbamidomethylDTT; L-methionine (S)-sulfoxide
Instrument	instrument model

Revision	Datetime	Status	ChangeLog Entry
0	2024-01-09 02:19:15	ID requested
⏵ 1	2024-01-12 14:27:15	announced

no publication

submitter keyword: bacteria, ribosome, quantification

Michel Arthur
contact affiliation	Centre de Recherche des Cordeliers, INSERM
contact email	michel.arthur@crc.jussieu.fr
lab head
rusconi
contact affiliation	CNRS
contact email	filippo.rusconi@universite-paris-saclay.fr
dataset submitter

MassIVE dataset URI

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