Reference Plasma Proteome Map of Crotalus durissus terrificus
Description
Little is known about the plasma proteome of snakes, a gap that limits physiological assessments and the development of species-specific reference protein profiles. This study aimed to characterize the plasma proteome of healthy adult Crotalus durissus terrificus snakes to establish a molecular baseline that supports biological evaluation and future diagnostic research.
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2D-SDS-PAGE protein profile was performed using a pooled plasma sample. In-gel digestion of each protein spot was performed following Shevchenko et al. (2006). Triptic peptides were recovered through sequential extractions using aqueous and organic solvents, pooled, and concentrated prior to LC–MS/MS analysis. Nano-LC-ESI-MS/MS analyses were conducted using a MicrOTOF-Q III Mass Spectrometer (Bruker Daltonics) coupled to an LC-20AT Liquid Chromatograph (Shimadzu) with binary pump system and autosampler. Samples were solubilized in 5% (v/v) acetonitrile/0.1% (v/v) TFA and peptides were loaded on C18 column (4.5 × 100 mm, 1.8 µm - Phenomenex) and eluted at a flow rate of 0.2mL/min using a linear gradient from Solution A [0.1% (v/v) TFA in water] to Solution B [100% acetonitrile with 0.1% (v/v) TFA], ranging from 5% to 80% of B over 60 min. Column temperature was monitored at 25°C and autosampler at 10°C. Mass spectrometry was performed in positive ionization at 4.5 kV, using data-dependent acquisition (DDA) mode, nitrogen flow at 6 L/min and 0.8 bar pressure. Mass spectra were acquired with a scan range of m/z 400–1500. For MS/MS, nitrogen was used as collision gas with 1–200 eV Energy. Internal and external calibration was performed with sodium formate, and data were processed using Bruker Data Analysis v3.3.
Institutions
- Universidade Estadual Paulista Julio de Mesquita Filho Faculdade de Medicina Campus de Botucatu