PXD025953

PXD025953 is an original dataset announced via ProteomeXchange.

Title	Proteomics analysis of mouse-derived bone marrow cells exposed to a lethal level of ionizing radiation
Description	The precipitated proteins from whole cells were resuspended in 50 μl of 500 mM ammonium bicarbonate containing 8 M urea, and reduced with 5 μl of 200 mM DTT. The samples were incubated at 90°C for 30 min and cooled to room temperature. Free cysteine residues were alkylated with 10 μl of 200 mM iodoacetamide for 60 min at room temperature in the dark, and the remaining iodoacetamide was quenched by adding 5 μl of 200 mM DTT. The samples were then mixed with 320 μl of 100 mM ammonium bicarbonate. The samples were incubated with 5 μg trypsin (AB Sciex) at 37°C for 18 h. The samples were desalted with C18 ZipTip (Millipore, Bedford, MA, USA) and eluted with H2O/acetonitrile (5/5; v/v). The ZipTip eluates were dried in a vacuum centrifuge. Desalted samples were rehydrated in 0.1% formic acid and analyzed with liquid chromatography (LC)-MS using a nanoLC Eksigent 400 system (Eksigent, AB Sciex), coupled online to a TripleTOF6600 mass spectrometer (AB Sciex). Peptide separation was performed using LC on a trap and elution configuration using a nano trap column (350 μm × 0.5 mm, 3 μm, 120 Å, AB Sciex) and a nano ChromXP C18 reverse-phase column (75 μm × 15 cm, 3 μm, 120 Å, AB Sciex) at 300 nl/min with a 90-min linear gradient of 8% to 30% acetonitrile in 0.1% formic acid, and then, with a 10-min linear gradient of 30% to 40% acetonitrile in 0.1% formic acid. The MS was operated in information-dependent acquisition mode, scanning full spectra (400–1500 m/z) for 250 ms, followed by up to 30 MS/MS scans (100–1800 m/z for 50 ms each), for a cycle time of 1.8 s. Candidate ions with a charge state between +2 and +5 and counts above a minimum threshold of 125 counts per second were isolated for fragmentation, and one MS/MS spectrum was collected before adding those ions to the exclusion list for 12 s. Rolling collision energy (CE) was used with a CE spread of 15. The MS was operated by Analyst TF 1.7.1 software (AB Sciex). For data-independent acquisition (SWATH acquisition), the parameters were set as follows: 100 ms TOF MS scan, followed by 200 variable SWATH windows each at a 50-ms accumulation time for m/z 400–1250. MS/MS SWATH scans were set at 5-Da windows that overlapped by 1 Da for m/z 400–1250 and varied on each side of the mass range. The total cycle time was 9.6 s. Rolling CE parameter script was used for automatically controlling the CE. Data Analysis—Acquired spectra were searched against the UniProt reviewed database using the Paragon algorithm embedded in ProteinPilot 5.0.1 software (AB Sciex), with the following search parameters: (i) sample type: identification, (ii) Cys alkylation: iodoacetamide, (iii) digestion: trypsin, (iv) instrument: TripleTOF 6600, (v) species: Mus musculus, (vi) ID focus: biological modifications, (vii) detected protein threshold: >0.05 (10% confidence). The detected protein threshold was set to the minimum level to enhance the number of wrong answers to enable the curve fitting by an independent false discovery rate (FDR) analysis45. This was carried out by the target-decoy approach provided with ProteinPilot software, which was used to assess the quality of the identifications. Identifications were considered positive when identified proteins and peptides reached a 1% local FDR46. The resulting .group file was loaded into Peakview (v2.2.0, AB Sciex), and peaks from SWATH runs were extracted with a peptide confidence threshold of 99% and a FDR <1%. The SWATH files were then exported to MarkerView software (version 1.3.0.1; AB Sciex), and peak areas of individual peptides were normalized to the sum of peak areas of all detected peptides. Statistics of PCA and OPLS-DA were performed using Simca software (Infocom Corp., Tokyo, Japan) for multivariate statistical analysis. Pareto scaling was applied to the peak area values acquired by SWATH prior to the analyses, which represents a compromise between the extremes of no scaling and unit variance scaling and involves dividing each spectral variable by the square root of its standard deviation after first centering.
HostingRepository	jPOST
AnnounceDate	2022-05-12
AnnouncementXML	Submission_2023-08-21_03:42:49.047.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Yota Tatara
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	S-carboxamidomethyl-L-cysteine; N6-acetyl-L-lysine; alpha-amino acetylated residue; 2-pyrrolidone-5-carboxylic acid (Gln); 2-pyrrolidone-5-carboxylic acid (Glu); L-methionine sulfoxide; phosphorylated L-histidine; O-phospho-L-serine; O-phospho-L-threonine; O4'-phospho-L-tyrosine
Instrument	TripleTOF 6600

Revision	Datetime	Status	ChangeLog Entry
0	2021-05-11 21:48:22	ID requested
1	2022-05-11 08:00:28	announced
2	2022-09-18 03:21:05	announced	2022-09-18: Updated FTP location.
⏵ 3	2023-08-21 03:42:49	announced	2023-08-21: Updated PubMed.

Tatara Y, Monzen S, Proteomics and secreted lipidomics of mouse-derived bone marrow cells exposed to a lethal level of ionizing radiation. Sci Rep, 13(1):8802(2023) [pubmed]

submitter keyword: mouse, bone marrow stromal cells, radiation

Satoru Monzen
lab head
Yota Tatara
contact affiliation	Hirosaki University
dataset submitter

jPOST dataset URI

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.jpostdb.org/JPST001169/