PXD079671

PXD079671 is an original dataset announced via ProteomeXchange.

Title	Crosslinking mass spectrometry data for Beta-Heavy-Spectrin segments 25-31 homodimerization
Description	Zero-length crosslinking was performed to identify crosslinked residues in Beta-Heavy-Spectrin segments 25-31 homodimers (BH25-31). Purified BH25-31 protein was dialyzed into PBS (10 mM NaH2PO4, 130 mM NaCl, pH 7.3), concentrated, and clarified before being crosslinked with 5 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2.5 mM N-hydroxysulfosuccinimide (sulfo-NHS) on ice in the dark for 1 hour. Crosslinking reactions were quenched by adding 20 mM dithiothreitol and then kept on ice for 20 additional minutes before adding an equal volume of 2x Laemmli buffer. Prior to separation by SDS-PAGE for mass spectrometry, samples were incubated at 37°C for 5 minutes. After gels were stained with NOVEX Colloidal Blue, proteins from excised bands were reduced, alkylated, and digested with trypsin, and analyzed by mass spectrometry at the Penn State College of Medicine Mass Spectrometry and Proteomics Core. Specifically, peptides were analyzed using the nanoElute UHPLC (Bruker Daltonics, Bremen, Germany) and the Bruker timsTOF fleX instrument (Bruker Daltonics, Bremen, Germany). Peptides were separated by reversed-phase chromatography using a trapping cartridge (Acclaim PepMap C18 100 Å, 5 mm x 0.3 mm, 160454; Thermo Fischer Scientific, Waltham MA) and the Bruker15 column (100 Å, 150 mm x 0.15 mm; Bruker Daltonics, Bremen, Germany). Mobile phase A was water with 0.1% formic acid, and mobile phase B contained acetonitrile with 0.1% formic acid. A gradient elution was performed at 0.5 uL/min with 0.1% formic acid with increasing concentrations of acetonitrile (2% at 0 sec, 26% at 1500 sec, and 32% at 1800 sec) in water. At 1806 sec, the flow rate was reduced to 1 uL/min with 95% acetonitrile with 0.1% formic acid. The source voltage was set to 1.4 kV with a dry gas flow of 3.0 L/min and dry temperature of 180°C. Data was acquired in data-dependent acquisition parallel accumulation serial fragmentation (DDA-PASEF) mode with MS1 set from 100-1700 m/z and MS2 set from 400-1000 m/z. The mobility range was 0.6-1.7 1/K­0 with 100 ms ramp and accumulation time. Calibration of the ion mobility dimension had been performed with an Agilent ion mix (622.0289, 0.9848; 922.0097, 1.1895; 1221.9906, 1.3820; Agilent Technologies, Santa Clara CA) spotted on a filter. Crosslinks were identified using pLINK (V2.3.11; cog-genomics.org), searching a database containing the sequence of BH25-31 and a database with common contaminants. Names of files correspond to the analyzed gel band (e.g. gel 1, lane 3, band C = G1_3C) as indicated by the annotated gel file.
HostingRepository	MassIVE
AnnounceDate	2026-08-02
AnnouncementXML	Submission_2026-08-02_12:02:54.246.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Chelsea Sarring
SpeciesList	scientific name: Drosophila melanogaster; common name: fruit fly; NCBI TaxID: 7227;
ModificationList	Oxidation; Carbamidomethyl
Instrument	timsTOF fleX

Revision	Datetime	Status	ChangeLog Entry
0	2026-06-13 10:39:46	ID requested
⏵ 1	2026-08-02 12:02:54	announced

no publication

submitter keyword: Crosslinking mass spectrometry, Spectrin, DatasetType:Proteomics

Claire Thomas
contact affiliation	The Pennsylvania State University
contact email	ClaireT@psu.edu
lab head
Chelsea Sarring
contact affiliation	The Pennsylvania State University
contact email	cxs1336@psu.edu
dataset submitter

MassIVE dataset URI

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