Computational protocol: Expression of an Androgenic Gland-Specific Insulin-Like Peptide during the Course of Prawn Sexual and Morphotypic Differentiation

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Protocol publication

[…] Tryptic peptides were purified on a C-18 column and dissolved in 0.1% formic acid. Nanoliquid chromatography and mass-spectrometry analysis was performed as described previously [] using a 75-μm internal diameter fused silica column, packed with C18 (New Objective, Woburn, MA, USA) connected to an Eksigent nano-LC system (Eksigent, Dublin, CA, USA). Mass spectra where acquired using an LTQ-ORBITRAP XL (Thermo Fisher Scientific, San Jose, CA, USA). Full MS and MS/MS fragmentation was performed in the data-dependent mode, which allows switching between MS and MS/MS analysis. After full MS acquisition in the orbitrap, the multiply charged top 6 abundant masses where chosen for CID MS/MS fragmentation in the LTQ under the following conditions: the CID fragmentation was performed at 35% collision energy and 30 ms activation time. Protein identification and validation were performed using Sequest and Mascot algorithms operated under Proteome Discoverer 1.0 (Thermo Fisher Scientific) using an unpublished database containing the Mr-IAG sequence, which allowed 0.8 Da and 10 ppm tolerance. […]

Pipeline specifications

Software tools Comet, Proteome Discoverer
Application MS-based untargeted proteomics
Organisms Homo sapiens, Macrobrachium rosenbergii