----------------- INFORMACIÓN GENERAL ------------------- 1. Título del dataset A new approach to study inflammation in fish: Serum proteinogram analysis in gilthead seabream (Sparus aurata) injected with λ-carrageenan 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: María Ángeles Esteban Institución: Universidad de Murcia Correo electrónico: aesteban@um.es ORCID: 0000-0002-6264-1458 Nombre: Francisco A. Guardiola Institución: Universidad de Murcia Correo electrónico: faguardiola@um.es ORCID: 0000-0002-1018-5446 3. Fecha de recogida de los datos (fecha única o rango de fechas): [21-02-2024---23-06-2024] 4. Fecha de depósito de los datos: [05-08-2024] 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 Specimens of gilthead seabream (100.38 g ± 1.49 g, 16.76 cm ± 0.12 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. Experimental design and blood collection Experiment 1. Acute inflammation: Twenty-four seabream specimens were randomly selected, anesthetized with clove oil (20 mg L-1, Guinama®) and intramuscularly injected in the left flank, beneath the lateral line at the level of the second dorsal fin. Two groups of fish (with two replicates per group) were established and the fish were injected with: i) 50 µL of phosphate-buffered saline (PBS, Fisher Bioreagents) (control group); and ii) 50 µL of λ-carrageenan (1%, Sigma-Aldrich) in PBS (final concentration = 0.5 mg fish-1). After 3, 6 and 24 h post-carrageenan injection (p.i.), three fish from each tank (n = 6 per group) were sedated as previously described, weighed and measured. Then, blood samples (approximately 100 µL) were collected from the caudal vein using an insulin syringe and the fish were returned to the aquaria considering the 3 Rs principle. Experiment 2. Chronic inflammation: Twelve seabream specimens were anesthetized and intramuscularly injected as previously described. Likewise, two groups of fish (with two replicates per group) were established and the fish were injected with: i) 500 µL of PBS (Fisher Bioreagents) (control group); and ii) 500 µL of λ-carrageenan (1%, Sigma-Aldrich) in PBS. Fish were injected again at 7 and 14 days with 250 µL of PBS (control) or 250 µL of λ-carrageenan (final concentration: 20 mg fish-1) to simulate a chronic inflammation disease with relapses. At 15 days p.i. three fish from each tank (n = 6 per group) were sedated, weighed and measured. Subsequently, blood samples were collected, and the fish were returned to the aquaria. 2.3. Serum protein concentration determination 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 ºC until use. Serum protein concentration was determined using an infrared-based protein quantitation method using 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 bovine serum albumin calibration curve method. 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 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 Samples were digested with the following standard procedure. Samples were dissolved in 100 µL of 50 mM ammonium bicarbonate buffer pH 8.5 with 0.01 % Protease Max (Promega). This surfactant enhances the trypsin digestion. Protein samples were reduced by adding 20 mM dithiothreitol (DTT) at 56 ºC for 20 min. Then, samples were alkylated by adding 100 mM indole-3-acetic acid (IAA) during 30 min at room temperature in the dark. Finally, digestion was performed by adding 1 µg of Trypsin Gold Proteomics Grade (Promega) (approx. 1:100 w/w) during 3 h at 37 ºC. Reaction was stopped with 0.1 % formic acid and filtered through 0.2 µm. Finally, samples were dried using an Eppendorf Vacuum Concentrator (model 5301). 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 (2.7 µm, 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 electropherograms 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. Figures were made with GraphPad 8 software. Results were expressed as mean ± standard error of the mean (SEM). Data from experiment 1 were analysed using Two-way-ANOVA to stablish significant differences between two factors: experimental group and time, and to determine the interaction of both factors. Data from experiment 2 were analysed by Student’s t-test to determine differences between experimental groups. The normality of the data was previously assessed using the Shapiro-Wilk test and the homogeneity of variance was also verified using Levene’s test. All statistical analyses were conducted using the computer package SPSS (28.0 version; SPSS Inc., Chicago, IL, USA) for Windows. The level of significance used was p < 0.05 for all statistical tests. 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: Computer package SPSS (28.0 version; SPSS Inc., Chicago, IL, USA) for Windows. 3. Procedimientos seguidos para asegurar la calidad de los datos Positive and negative controls in each assay ------------------------ 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 inflammation_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.]