PXD068969

PXD068969 is an original dataset announced via ProteomeXchange.

Title	Proteomics analysis of Hydractinia symbiolongicarpus nematocysts
Description	Hydractinia symbiolongicarpus nematocysts were sorted from ca. 120 polyps that had been dissociated in 1% pronase. Two gating strategies were used, a large gate (LG) and a more conservative small gating (SG) strategy to limit the number of dead cells. About 300,000 nematocysts were obtained for both sorting strategies, flash-frozen, and stored at -70C until further use. Nematocysts were lysed through freeze/thaw/sonicate cycles. The tubes were centrifuged at 14,000g for 10 min, and the supernatant was dried using a SpeedVac concentrator (Savant) without heat. The dried protein pellets were stored at -20C. Proteins were solubilized in 30 ul of 8M urea,100mM Tris, pH8.5, vortexed, and sonicated 10 times with 30 sec ON/30 sec OFF cycles in a water bath at RT. Proteins were reduced by adding TCEP at 1M to 5 mM final at RT for 30 min. Reduced cysteine residues were carboxymethylated by adding 5 ul of 0.5M CAM and samples were incubated for 30 min in the dark at RT. Endoproteinase Lys-C at 0.1ug/ul was added at 1:1000 w/w and the digestion proceeded for over 6 hrs at 37C. The sample was diluted to 2M urea with 100mM Tris, pH8.5, 2mM CaCl2, and sequencing grade modified trypsin was added at 1:200 w/w, at 37C O/N. Peptide amounts calculated from a fluorometric assay were ca. 400 and 200 ng for the LG and SG samples, respectively. Peptides were dried using a SpeedVac concentrator. The dried peptide mixture was resuspended in 300 ul of 0.1% trifluoroacetic acid (TFA) and loaded onto one high pH fractionation cartridge placed on a new 2 ml sample tube. After centrifuging at 3000g for 2 min, the eluate was collected as the "flow-through" fraction. The loaded cartridge was placed on a new 2 ml sample tube, washed with 300 ul of ddH20, and the eluate collected as "wash" fraction. A total of 8 HpH RP fractions were collected by sequential elution in new sample tubes using 300 ul of 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, and 50% acetonitrile in 0.1% TFA. Peptides were analyzed on an Orbitrap Eclipse Tribrid Mass Spectrometer with a FAIMS Pro interface, equipped with a Nanospray Flex Ion Source, and coupled to a Dionex UltiMate 3000 RSCLnano System (U3000). A 75 um i.d. analytical microcapillary column was packed in-house with 250 mm of 1.9 um ReproSil-Pur C18-AQ resin (Dr. Masch). AgileSLEEVE was used to maintain column temperature at 50C. Peptides from each of the 8 HpH RP fractions were solubilized in 22 ul of buffer A (5% acetonitrile in 0.1% formic acid, FA) and loaded via a 20 uL sample loop on an Acclaim PepMap 100 C18 trap cartridge (0.3 mm inner diameter (i.d.), 5 mm length; Thermo Fisher Scientific) with the U3000 loading pump at 2 uL/minute via the autosampler. The organic solvent solutions were water:acetonitrile:formic acid at 95:5:0.1 (volume ratio) for buffer A (pH 2.6) and 20:80:0.1 (volume ratio) for buffer B. Peptides were eluted directly into the mass spectrometer over a 95-min chromatography gradient with a nano pump flow rate set to 0.180 ul/min. The Orbitrap Eclipse was set up to run the MS OT/ddMS2 IT HCD method with two FAIMS CVs at -40V and -60V and a cycle time of 1 sec. Briefly, eluting peptide ions were scanned from 375-1500 m/z in the Orbitrap at 120,000 resolving power before ddMS2 fragmentation by HCD at 35% NCE and detection in the ion trap set to rapid scan rate. The isolation window was 1.6 m/z and dynamic exclusion, with low and high mass tolerances set at 10 ppm, was enabled for 45s.
HostingRepository	MassIVE
AnnounceDate	2026-02-05
AnnouncementXML	Submission_2026-02-05_08:54:09.229.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Stowers Proteomics
SpeciesList	scientific name: Hydractinia symbiolongicarpus; NCBI TaxID: 13093;
ModificationList	L-methionine sulfoxide; S-carboxamidomethyl-L-cysteine
Instrument	Orbitrap Eclipse

Revision	Datetime	Status	ChangeLog Entry
0	2025-09-29 10:39:54	ID requested
⏵ 1	2026-02-05 08:54:09	announced

no publication

submitter keyword: cnidarians, stinging cells, DatasetType:Proteomics

Laurence Florens
contact affiliation	The Stowers Institute for Medical Research
contact email	laf@stowers.org
lab head
Stowers Proteomics
contact affiliation	Stowers Institute for Med Res
contact email	proteomicslab@stowers.org
dataset submitter

MassIVE dataset URI

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