The analysis of the obtained spectra was done using ProteinPilot v 4.5. Search engine (ABSciex). ProteinPilot default parameters were used to generate a peak list directly from the 5600 TripleTof wiff files. The Paragon algorithm (Shilov et al., 2007) (link) of ProteinPilot v 4.5 was used to search the Uniprot_Aves (Nov 2018) database using none enzyme as parameter. The posttranslational modifications such as oxidation of methionine and proline amino acids were determined automatically by Paragon algorithm. Samples were grouped by type for the search (CN and CPF). Additionally, all samples were combined in a single search to build the library for quantitation. The protein grouping was done by Pro group algorithm. Peak View 1.1 software (AB Sciex, Framingham, MA, USA) was used to quantify the areas for all the peptides assigned in the library. Only peptides with confidence of 95% or greater were quantified. The areas obtained previously were loaded on Marker View 1.3 software (AB Sciex, Framingham, MA, USA) and data were normalized by total areas sum. Normalized areas were used to extract the information of oxidized peptides, which were used for statistics analysis. Principal Component Analysis (PCA) and loading plot analysis were performed using SIMCA-P+ 13.0 software (Umetrics AB, Sweden).
Peakview1
Manufactured by AB Sciex
Sourced in United States
The PeakView1.2 is a high-performance liquid chromatography system produced by AB Sciex. It is designed for the separation and analysis of complex samples. The PeakView1.2 provides precise control over solvent flow, temperature, and other parameters to ensure accurate and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Markerview 1, by AB Sciex (4 mentions)
Tripletof 5600, by AB Sciex (4 mentions)
Multiquant 2, by AB Sciex (3 mentions)
Simca p 13, by Sartorius (2 mentions)
Duo spray source, by AB Sciex (2 mentions)
Tripletof 5600 mass spectrometer, by AB Sciex (2 mentions)
Isopropyl alcohol, by Thermo Fisher Scientific (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Eksigent lc100, by AB Sciex (2 mentions)
Xbridge peptide beh c18 column, by Waters Corporation (2 mentions)
Mascot distiller v2, by Matrix Science (1 mentions)
Sil 30acxr, by Shimadzu (1 mentions)
Uhplc system, by Shimadzu (1 mentions)
Sil 30acxr autosampler, by Shimadzu (1 mentions)
Zorbax eclipse plus c18 column, by Agilent Technologies (1 mentions)
Triple tof 5600 system, by AB Sciex (1 mentions)
Thermo xcalibur 2, by Thermo Fisher Scientific (1 mentions)
Prism software, by GraphPad (1 mentions)
Peaks studio 10, by Bioinformatics Solutions (1 mentions)
Multiquant 3, by AB Sciex (1 mentions)
24 protocols using peakview1
1
Mass Spectrometry-based Proteomic Analysis
2
Label-free Quantitative Proteomics Workflow
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Relative quantitation was performed using a label-free method according to Gallego et al. (2015a) . The quantitation of peptide is based on the relative integrate area of extracted ion chromatogram (XIC) in each sample. For that, quantitative data was obtained using Peak View 1.1 software (AB Sciex, Framingham, MA, USA) and then analysed using Marker View 1.3 software (AB Sciex, Framingham, MA, USA).
No multiple corrections are performed. Only peptides with confidence of 95% or greater were quantified. The area of extracted ion chromatograms (XICs) for each peptide was obtained from the median according to the predefined algorithm in Peak View 1.1 software. Each sample was quantified in triplicate.
No multiple corrections are performed. Only peptides with confidence of 95% or greater were quantified. The area of extracted ion chromatograms (XICs) for each peptide was obtained from the median according to the predefined algorithm in Peak View 1.1 software. Each sample was quantified in triplicate.
3
Label-free Protein Quantification Protocol
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The identification of protein origin of peptides was done using using Mascot Distiller v2.4.2.0 software (Matrix Science, Inc., Boston, MA). The search parameters were established as: Expasy database under Mammalia taxonomy, significance threshold of p<0.05 and a FDR of 1.5%, and a tolerance of 100 ppm in MS mode and 0.3 Da for MS/MS. Oxidation of Methionine was considered as variable modification and none specific enzyme was selected.
The relative quantification was done using the label-free methodology described by Gallego, Mora, Aristoy and Toldrá, (2016) with small modifications. The analysis is divided into different steps: peak list generation, search in databases for identification of peptides, alignment of eXtracted Ion Chromatograms (XICs), and combination of the obtained areas to determine the ratios for individual peptides. A total of 18 samples were analysed and peptides were quantified using Peak View 1.1 software (AB Sciex, Framingham, MA, USA). The quantitation data obtained by Peak View was analysed with Marker View 1.2 (AB Sciex, Framingham, MA, USA) and areas were normalised by total areas summa, and Principal Component Analysis (PCA) analysis and T-test were done.
The relative quantification was done using the label-free methodology described by Gallego, Mora, Aristoy and Toldrá, (2016) with small modifications. The analysis is divided into different steps: peak list generation, search in databases for identification of peptides, alignment of eXtracted Ion Chromatograms (XICs), and combination of the obtained areas to determine the ratios for individual peptides. A total of 18 samples were analysed and peptides were quantified using Peak View 1.1 software (AB Sciex, Framingham, MA, USA). The quantitation data obtained by Peak View was analysed with Marker View 1.2 (AB Sciex, Framingham, MA, USA) and areas were normalised by total areas summa, and Principal Component Analysis (PCA) analysis and T-test were done.
4
Label-free Proteomics Workflow for Protein Quantification
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Data were processed using ProteinPilot TM v4.5 software (AB Sciex, MA, USA) for the identification and quantification of the peptides. The Paragon algorithm of ProteinPilot was used to search in ExPASy database (http://www.expasy.org) with no enzyme specificity and no cysteine alkylation. A novel database from Uniprot including all proteins from Sus scrofa species, and specifically titin protein from Mus musculus species and LIM domain binding protein 3 (LDB3) from Homo sapiens species (accession numbers A2ASS6 and O75112, respectively) was generated for the search.
A label-free methodology was used for the relative quantification of peptides, following the methodology described by Gallego et al. (2015a) . In this label-free approach, quantification was done at peptide level based on the measurement of the integrated areas of extracted ion chromatograms (XICs). Thus, the combination of XICs allows the determination of the ratios for individual peptides, using 3 replicates per sample and normalising by total areas summary. Peptides were quantified using Peak View 1.1 software (AB Sciex, Framingham, MA, USA) and then data were statistically analysed using Marker View 1.3 software (AB Sciex, Framingham, MA, USA). Principal Component Analysis (PCA) and loading plot analysis were performed using SIMCA-P+ 13.0 software (Umetrics AB, Sweden).
A label-free methodology was used for the relative quantification of peptides, following the methodology described by Gallego et al. (2015a) . In this label-free approach, quantification was done at peptide level based on the measurement of the integrated areas of extracted ion chromatograms (XICs). Thus, the combination of XICs allows the determination of the ratios for individual peptides, using 3 replicates per sample and normalising by total areas summary. Peptides were quantified using Peak View 1.1 software (AB Sciex, Framingham, MA, USA) and then data were statistically analysed using Marker View 1.3 software (AB Sciex, Framingham, MA, USA). Principal Component Analysis (PCA) and loading plot analysis were performed using SIMCA-P+ 13.0 software (Umetrics AB, Sweden).
5
UHPLC-MS Analysis of Metabolites
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Analyses of all samples were performed on a UHPLC system (Shimadzu, Kyoto, Japan) which is composed of a solvent delivery system LC-3AD, an autosampler SIL30ACXR, a column oven CTO-30AC, a degasser DGU-20A3, and a controller CBM-20A. The separation was conducted on an UHPLC Agilent column Zorbax EcLipse Plus C18 at a flow rate of 0.3 mL/min (1.8 μm, 100 mm × 2.1 mm). During the analysis, the column temperature was maintained at 40 °C. The mobile phase is made of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The elution program was set as follows: 0–15 min, 10%–95% B; 15.01–19 min, 10% B. A triple TOFM X500R system equipped with a Duo Spray source (AB SCIEX, Foster City, CA, USA) was used to conduct the MS analysis in the negative electrospray ion mode. The parameters of electrospray ionization (ESI) were set as follows: ion spray voltage: −4500 V; curtain gas: 35 psi; ion source temperature: 550 °C; declustering potential (DP): −100 V, nebulizer gas (GS 1): 55 psi, and heater gas (GS 2): 55 psi. m/z 100–1600 Da was used for the MS scan. Data analysis were performed by the Peak View® 1.4 software (AB SCIEX Foster City, CA, USA).
6
UHPLC-QTOF-MS/MS Analysis of Plant Extracts
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Analyses of all the samples, including the ACB total extract, ES, EA, EB and IEA, were carried out on a Shimadzu system (Kyoto, Japan) equipped with an LC-3AD solvent delivery system, a SIL-30ACXR auto-sampler, a CTO-30AC column oven, a DGU-20A3 degasser, and a CBM-20A controller. All samples were separated on an Agilent Zorbax Eclipse Plus C18 column (100 mm×2.1 mm, 1.8 µm, flow rate: 0.35 mL/min). The column oven temperature was set at 40°C. The mobile phase consisted of A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile): 0–8 min, 5–70% B; 8–11 min, 70–100% B; 11–12 min, 100% B; 12–15 min, 5% B.
The UHPLC-QTOF-MS/MS analysis was conducted on a triple TOF™ X500R system with a Duo Spray source in the negative electrospray ion mode (AB SCIEX, Foster City, CA, USA). The electrospray ionization was applied in the negative mode with the following parameters: ion spray voltage, –4,500 V; ion source temperature, 500°C; curtain gas, 25 psi; nebulizer gas (GS 1), 50 psi; heater gas (GS 2), 50 psi; and declustering potential (DP), –100 V. The mass ranges were set at m/z 60 –2,000 Da for the TOF-MS scan. The LC-MS/MS data were analyzed using the Peak View® 1.4 software (AB SCIEX Foster City, CA, USA).
The UHPLC-QTOF-MS/MS analysis was conducted on a triple TOF™ X500R system with a Duo Spray source in the negative electrospray ion mode (AB SCIEX, Foster City, CA, USA). The electrospray ionization was applied in the negative mode with the following parameters: ion spray voltage, –4,500 V; ion source temperature, 500°C; curtain gas, 25 psi; nebulizer gas (GS 1), 50 psi; heater gas (GS 2), 50 psi; and declustering potential (DP), –100 V. The mass ranges were set at m/z 60 –2,000 Da for the TOF-MS scan. The LC-MS/MS data were analyzed using the Peak View® 1.4 software (AB SCIEX Foster City, CA, USA).
7
Quantitative Proteomics Analysis of Enzyme
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The LC-MS system consisted of a NanoLC Ultra System (Eksigent Technologies, Dublin, CA, USA), a TripleTOF 5600 System (AB SCIEX, Framingham, MA, USA) equipped with a trap column (150 μm I.D. × 10 mm L.; C18, 3 μm, 100 Å; Proteomics Front, Beijing, China) and a separation column (75 μm I.D. × 150 mm L., C18, 3 μm, 100 Å; Proteomics Front). The dried peptides were dissolved in 20 μl of solvent A [5% ACN, 0.1% Formic Acid (FA)], and a 5-μl aliquot was injected into the trap column at a flow rate of 2.0 μl/min. The analytical separation was conducted at a flow rate of 300 nl/min. After a 4-min wash with 5% solvent B (95% ACN, 0.1% FA), a 45-min linear gradient was run with 5–80% solvent B followed by a 5-min linear gradient with 80–95% solvent B. The eluate was directly evaporated at 150 °C with a nitrogen stream of 3 l/min, and the ion spray voltage was set at 2.3 kV. MS was operated in the positive-ion mode with a mass range of 400–4000 m/z. MS/MS was acquired in an automated data-dependent acquisition mode with charge numbers of 2–4 and a mass range of 100–2000 m/z. The data were analyzed with PeakView 1.2 software (AB SCIEX). The sequence coverages were analyzed with ProteinPilotTM software using Database uniprot_sprot_20121010 specifically towards Accession # sp|P21163|PNGF_ELIMR54 (link).
8
Compound Identification via Mass Spectrometry
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Generally, common characteristic peaks were identified by comparison with the reference compounds. If no standard reference was available for the compound, the characteristic peaks were inferred using Peakview 1.2 software (AB SCIEX, Framingham, MA, USA) and online resources, such as SciFinder (https://scifinder.cas.org/ (accessed on 13 May 2022)), HMDB (https://hmdb.ca/ (accessed on 20 May 2022)) and CNKI (https://kns.cnki.net/ (accessed on23 May 2022)) by comparing MS/MS fragment ions.
9
Metabolomics Analysis by UHPLC-Q-TOF-MS/MS
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The data collected via UHPLC-Q-TOF-MS/MS were processed using Peak View 1.2 software (AB SCIEX, version 1.2.0.3) from AB Sciex Company. The empirical molecular formulae were deduced from Peakview by comparing the theoretical masses of molecular ions and/or adductions with the determined values based on the following error limits: mass accuracy, < 5 ppm; retention time, < 5.0%; and isotope abundance, < 10%. The multistage mass spectrometry data collected via ESI-LTQ-Orbitrap-MSn were processed using Thermo Xcalibur 2.2 (Thermo Fisher Scientific).
10
Characterization of hpMR1 Molecules
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