----------------- INFORMACIÓN GENERAL ------------------- 1. Título del dataset Serum proteinogram of gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) as a new useful approach for detecting loss of haemostasis 2. Autoría: [Rellenar la información de todos los autores siguiendo el siguiente formato. Repetir el esquema, uno para cada autor.] Nombre: Jose Carlos Campos-Sánchez Institución: Universidad de Murcia Correo electrónico: josecarlos.campos@um.es ORCID: 0000-0003-0303-5412 Nombre: Francisco A. Guardiola Institución: Universidad de Murcia Correo electrónico: faguardiola@um.es ORCID: 0000-0002-1018-5446 Nombre: María Ángeles Esteban Institución: Universidad de Murcia Correo electrónico: aesteban@um.es ORCID: 0000-0002-6264-1458 3. Fecha de recogida de los datos (fecha única o rango de fechas): [09-07-2022---18-01-2024] 4. Fecha de depósito de los datos: [03-14-2024] 5. Idioma del conjunto de datos: Inglés ------------------------ INFORMACIÓN METODOLÓGICA ------------------------ 1.Descripción de la metodología utilizada para generar el conjunto de datos. 2. Material and methods 2.1. Animals Fifty healthy and independent fish specimens of gilthead seabream (S. aurata) (43.60 g ± 4.92 g, 13.18 cm ± 0.39 cm) and fifty of European seabass (D. labrax) (40.40 g ± 3.07 g, 15.67 cm ± 0.38 cm) were obtained from a local farm (Murcia, Spain) and kept in re-circulating seawater aquaria (450 L) at the Marine Fish Facilities at the University of Murcia (Spain) during a quarantine period of one month. Water temperature was maintained at 20 ± 2 °C with a flow rate of 900 L h−1, a salinity of 28‰, an artificial photoperiod of 12 h light to 12 h dark and with continuous aeration. Water ammonium and nitrite levels were maintained below the limits for the species (0.1 mg L−1 and 0.2 mg L−1, respectively). Fish were fed with a commercial diet (Skretting, Spain) at a rate of 2 % body weight day−1 and were kept 24 h without feeding before sampling. Fish maintenance, experimental procedures and euthanasia were carried out in compliance with ethical standards, and the protocol was approved by the Ethical Committee for Animal Experimentation of the University of Murcia (permit number A13160416), adhering to the European directive 2010/63/EU on the protection of animals used for scientific purposes. 2.2 Serum obtention and protein concentration determination Ten fish from each of the two fish species were randomly selected and anesthetized using clove oil (20 mg L-1, Guinama®). After anesthetizing the fish, blood samples were collected from the caudal vein using an insulin syringe. The collected blood samples were allowed to clot at 4 °C for 4 h, and the serum was collected after centrifuging the samples (10,000 x g, 5 min, 4 °C). The collected serum was stored at -20 °C until it was processed. The protein concentration of the serum was determined using an infrared-based protein quantitation method with a Direct Detect Spectrometer (Merck-Millipore). The serum samples were diluted 10 times in PBS, and aliquots of 2 µL were spotted on the measuring card, while PBS was used as a reference buffer. The measuring was done using the standard BSA calibration curve method. 2.3. Protein separation Protein separation was carried out in a standard home-made 12 % minigel (8 cm x 8 cm x 1 mm thick) using a Biorad MiniProtean TetraCell system with denaturing conditions (polyacrylamide gel electrophoresis with sodium dodecyl sulphate; SDS-PAGE). Running buffer composition was 25 mM Tris-HCl pH 8.3, 192 mM glycine and 0.1% (w/V) SDS. A prestained molecular weight marker (Pageruler Plus prestained protein ladder, Thermofisher Scientific) was added in one well of the gel. Briefly, 10 µL of each diluted serum sample were added to the gel, and electrophoresis was completed applying 150 V for 20 min and then 200 V for 30 min (until the front reached the bottom of the gel). Gels were stained with a commercial Blue coomassie solution (Pageblue from ThermoScientific) according to the manufacturer´s recommendations and washed with 200 mL MilliQ water for 10 min three times in square dish plates (24.5 x 24.5 cm) before being incubated with this solution (1 h, room temperature). Gels were washed repeatedly with MilliQ water. An orbital shaker was used at a low speed (30 to 50 rpm). 2.4. Electropherogram Based on the data obtained from infrared protein quantification, the serum samples were diluted in Milli-Q water and adjusted to 1,500 ng µL-1. Protein sizing and quantification were then performed using the Agilent Protein80 and Protein230 kits on the Agilent 2100 Bioanalyzer (Agilent Technologies) according to the manufacturer's instructions. Data analysis was performed using Agilent 2100 Expert software. Size estimation compared peak migration times to an external size standard (ladder), whereas concentration estimation compared peak areas to internal standard proteins (low and high labels). 2.5. High performance liquid chromatography- Mass spectrometry The separation and analysis of the tryptic digests of the diluted serum samples were performed with a HPLC/MS system consisting of an Agilent 1290 Infinity II Series HPLC (Agilent Technologies) equipped with an Automated Multisampler module and a High Speed Binary Pump, and connected to an Agilent 6550 Q-TOF Mass Spectrometer (Agilent Technologies) using an Agilent Jet Stream Dual electrospray (AJS-Dual ESI) interface. Experimental parameters for HPLC and Q-TOF were set in MassHunter Workstation Data Acquisition software (Agilent Technologies, Rev. B.08.00). Dry samples from trypsin digestion were resuspended in 20 µL of buffer A, consisting in water/acetonitrile/formic acid (94.9:5:0.1). The samples were injected onto an Agilent AdvanceBio Peptide Mapping HPLC column (2.7µm, 100 x 2.1 mm, Agilent Technologies), thermostatted at 50 C, at a flow rate of 0.4 mL min-1. This column is suitable for peptide separation and analysis. After the injection, the columns were washed with buffer A for 3 min and the digested peptides were eluted using a linear gradient 0-40 % B (buffer B consisting of water/acetonitrile/formic acid, 10:89.9:0.1) for 40 min followed by a linear gradient 40-95 % B for 8 min and a 95% B for 3 min. Finally, the columns were equilibrated in the initial conditions for 6 min before every injection. The mass spectrometer was operated in the positive mode. The nebulizer gas pressure was set to 35 psi, whereas the drying gas flow was set to 14 L min-1 at 300 C, and the sheath gas flow was set to 11 L min-1 at 250 ºC. The capillary spray, nozzle, fragmentor and octopole RF Vpp voltages were 3500 V, 100 V, 360 V and 750 V, respectively. Profile data were acquired for both MS and MS/MS scans in extended dynamic range mode at 4 GHz. MS and MS/MS mass range was 50-1700 m z-1 and scan rates were 8 spectra sec-1 for MS and 3 spectra sec-1 for MS/MS. Auto MS/MS mode was used with precursor selection by abundance and a maximum of 20 precursors selected per cycle. A ramped collision energy was used with a slope of 3.68 and an offset of -4.28. The same ion was rejected after two consecutive spectra. The Spectrum Mill MS Proteomics Workbench (Rev B.06.00.201, Agilent Technologies, Santa Clara, CA, USA) was used for data processing and analysis. Briefly, raw data were extracted under default conditions as follows: unmodified or carbamidomethylated cysteines; [MH]+50-10000 m/z; maximum precursor charge +5; minimum signal-to-noise MS (S/N) 25; search for 12C signals. The MS/MS search against the corresponding and updated protein database was performed with the following criteria Variable modification search mode (carbamidomethylcysteine, STY phosphorylation, oxidation of methionine, and conversion of N-terminal glutamine to pyroglutamic acid); tryptic digestion with 5 maximum missed cleavages; ESI-Q-TOF instrument; minimum matched peak intensity 50%; maximum ambiguous precursor charge +5; monoisotopic masses; peptide precursor mass tolerance 20 ppm; product ion mass tolerance 50 ppm; and reverse database score calculation. Automated thresholds were used to validate peptide and protein data. 2.6. Data analysis The protein concentration data obtained from the electropherogram were classified according to their molecular weight. Proteins with a single peak in the electropherogram were directly averaged between individuals. Proteins with two or more peaks in an individual were summed and then the mean was calculated. The final data were obtained by taking the sum of all the concentrations as 100% and then adjusting the values accordingly. The ranges of each protein fraction were calculated by considering the lower and upper limits of the major protein it contained. 2. Software o instrumentos necesarios para interpretar los datos: [Incluir la versión del software. Si hace falta un software específico de acceso restringido, explicar cómo obtenerlo. Valorar si es posible cambiar el conjunto de datos a un formato abierto (recomendado).] Data analysis: Microsoft Excel or similar. 3. Procedimientos seguidos para asegurar la calidad de los datos Standards and markers were used to determinate size and concentration of proteins. ------------------------ ESCTRUCTURA DE LOS ARCHIVOS --------------------------- 1. Nombres de archivos [Mencionar todos los archivos incluidos en el conjunto de datos, con el nombre y la extensión (.csv, .pdf, etc.) de cada fichero]. Proteinogram - Raw data.xls 2. Formato de los archivos: Libro de Excel 97-2003 (*.xls) ------------------------ MÁS INFORMACIÓN ------------------------ [Incluir cualquier otra información sobre el conjunto de datos que no haya quedado reflejada en esta plantilla y que se considere relevante.]