Northern elephant seal outer blubber layer proteome
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Mirounga Angustirostris (ncbitaxon:9716)
SUBMITTER: 
Jane Khudyakov
PROVIDER: MSV000082455 | MassIVE | Tue Jun 12 17:13:00 BST 2018
REPOSITORIES: MassIVE
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Three injections were used for each sample. For each run, 5 uL of the sample (1 ug total) was loop injected onto a reversed-phase trap column (Acclaim PepMap 100 C18 LC column; 75 um i.d. x 2 cm, 3 um particle size, 100 A pore size, Thermo Fisher Scientific, USA) by a Dionex Ultimate 3000 autosampler. Peptides were eluted onto a reversed-phase analytical column set at 35C for HPLC (EASY-SprayTM C18 LC column; 75 um i.d. x 15 cm, 100 A, Thermo Fisher Scientific, USA). Solvent A was water and B was acetonitrile (ACN), respectively (both with 0.1 percent formic acid). During each chromatographic run, which lasted 140 min, flow rates were held at 300 nl/min. Solvent B (ACN) was used as follows: 2 percent at 5 min, 2-22 percent at 75 min, 22-38 percent at 100 min, 38-95 percent at 105 min, 95 percent returning to 2 percent at 115 min, and lastly maintained at 2 percent at 140 min.
Mass spectrometry analysis was performed using Orbitrap Fusion Tribrid mass spectrometer equipped with an EASY-SprayTM ion source and operated by Xcalibur 4.0 software. MS1 spectra were resolved by the orbitrap with a resolution of 120,000, scan range of 200-1400 m/z, RF lens of 60 percent, AGC target of 1.0e6, and max injection time of 50 ms. Precursor ions were isolated by quadrupole and fragmented using HCD with a collision energy of 28 percent. MS2 product ions were resolved by the orbitrap (resolution: 30,000, AGC target: 5.0e5, first mass: 100 m/z, and max injection time: 150 ms).
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Project description:Washed three times in NaCl (150 mM). Cells were resuspended in 1 ml TSU buffer (50 mM Tris pH 8.0, 0.1% SDS, 2.5 M urea) and lysed by two freeze-thaw cycles in liquid nitrogen. Lysate proteins (40 ug) were separated and digested. Digested peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex; Amsterdam, Netherlands). Samples (1 ul) were concentrated and desalted onto a micro C18 pre-column (500 um×2 mm, Michrom Bioresources; Auburn, CA, USA) with H2O:CH3CN (98:2, 0.05% trifluoroacetic acid) at 15 ul min−1. After a 4 min wash the pre-column was switched (Valco 10 port valve; Dionex) into line with a fritless nano column (75 u×~10 cm) containing C18 media (5 u, 200 A Magic; Michrom) manufactured according to Gatlin. Peptides were eluted using a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (64:36, 0.1% formic acid) at 250 nl min−1 over 30 min. High voltage (2000 V) was applied to low volume tee (Upchurch Scientific) and the column tip positioned ~0.5 cm from the heated capillary (T = 280 degrees C) of an Orbitrap Velos (Thermo Electron; Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray and the Orbitrap operated in data dependent acquisition mode (DDA). A survey scan m/z 350–1750 was acquired in the Orbitrap (Resolution = 30,000 at m/z 400, with an accumulation target value of 1,000,000 ions) with lockmass enabled. Up to the 10 most abundant ions (>5,000 counts) with charge states >+2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q = 0.25 and activation time of 30 ms at a target value of 30,000 ions. M/z ratios selected for MS/MS were dynamically excluded for 30 s. Peak lists were generated using Mascot Daemon/extract_msn (Matrix Science, Thermo; London, England) using the default parameters, and submitted to the database search program Mascot (version 2.1, Matrix Science). Search parameters were: Precursor tolerance 4 ppm and product ion tolerances +/-0.4 Da Oxidation (M) and Carbamidomethyl (C) specified as variable modifications Enzyme specificity was trypsin, 1 missed cleavage was possible
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