DOI: 10.25345/c5b853x13
title: MassIVE MSV000099316 - Proteomics analysis of Hydractinia symbiolongicarpus nematocysts
title: 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.