Ltq orbitrap xl etd

Heat soluble proteomics was conducted as previously described [30 (link)]. Briefly, approximately 3000 individuals were collected from the wild and cleaned as described above and homogenized using BioMasher II (Nippi) in PBS (Nippon Gene) on ice with protease inhibitors (Roche). The lysate was heated at 92 °C for 20 min, and the soluble fraction was collected by taking the supernatant after centrifugation at 12,000 rpm for 20 min. Proteins were digested with trypsin, and tryptic peptides were separated and identified with an UltiMate 3000 nanoLC pump (Dionex Co., Sunnyvale, CA, USA) and an LTQ Orbitrap XL ETD (Thermo Electron, Waltham, MA, USA). Corresponding peptide sequences were retrieved from six frame translation data of our initial genome assembly using MASCOT software (Matrix Science) [79 (link)]. Candidates were further screened with the following conditions: (1) lack of conservation in other tardigrades or metazoans and (2) high mRNA expression (TPM > 100) in the tun state. We then predicted the structural features using the Fold Index [80 (link)] and DISOPRED [81 (link)].

Each sample for proteome analysis was dissolved with 12 µL of 0.5% acetic acid 5% acetonitrile, and 5 µL of the solution was loaded on hand-made spray needle column (Reprosil-Pur C18 materials, 100 µm i.d. Dr. Maisch GmbH, Germany, 5 µm tip i.d., 130 mm length) using a HTC-PAL autosampler (CTC Analytics, Zwingen, Switzerland). The peptide fragments in the samples were separated through the column by reversed phase chromatography of linear gradient mode using UltiMate 3000 nanoLC Pump (Dionex Co., Sunnyvale, CA, USA). As the mobile phases, (A) acetic acid/water (0.5:100, v/v), (B) acetic acid/acetonitrile (0.5:100, v/v) and (C) acetic acid/dimethyl sulfoxide (0.5:100, v/v) were mixed keeping the flow rate of 500 nL/min. The composition was changed as follow: (A) + (B) = 96%, (C) = 4%, (B) 0–4% (0–5 min), 4–24% (5–65 min), 24–76% (65–70 min), 76% (70–80 min), and 0% (80.1–120 min). The separated peptides were ionized at 2400 V by positive electrospray method, injected into LTQ orbitrap XL ETD (Thermo Electron, San Jose, CA, USA) and detected as peptide ions (scan range: m/z300–1500, mass resolution: 60000 at m/z 400). Top 10 peaks of multiple charged peptide ions were subjected to collision-induced dissociation (isolation width: 2, normalized collision energy: 35 V, activation Q: 0.25, activation time: 30 s) to identify the amino acid sequence.

LC-ESI-MS and LC-ESI-MS-MS analyses were done on a LTQ Orbitrap XL ETD mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with standard ESI ion source. 5 μL of sample was injected at a flow rate of 50 μL/min in 80% ACN/H₂O with 0.1% FA by Ultimate 3000 RSLC system from Dionex (Dionex Corporation, Sunnyvale, CA). The conditions for full-scan MS are as follows: mass range m/z 0-6000 and resolution 60,000 at m/z 400. The target ions were sequentially isolated for MS2 by LTQ. Electrospray voltage was maintained at 4 kV and capillary temperature was set at 275 °C.

The freshly thawed samples were treated with deionized water or normal saline directly before the analysis. All samples were analyzed using the ICE technique and an LTQ Orbitrap XL ETD (ThermoFisher Scientific, San Jose, CA, USA) mass spectrometer as described elsewhere [14 (link)]. Briefly, the sample was placed inside a lab-made disposable cartridge (made from consumables provided by IDEX Health & Science LLC., Oak Harbor, WA, USA and GE Healthcare, Chicago, IL, USA) attached to the ESI interface of a mass spectrometer. The solvent flow (90% v/v methanol, 0.1% v/v acetic acid) extracts the analytes from the samples and delivers them into the ion source, while a glass microfiber filter inside the cartage protects the ESI needle from clogging. Mass spectra were acquired at the FTMS mass resolution set at 30,000 (FWHM at m/z 400 Da) for m/z values ranging from 500 to 1000 in the negative and positive modes, consecutively (30 s per polarity, 16 segments total). After the acquisition the ion source was washed with the solvent until the intensity of the signal decreased by at least 2 orders of magnitude from the level at the end of acquisition to avoid cross-contamination.