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Spectronaut pulsar x software

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Spectronaut Pulsar X is a software platform for data analysis in mass spectrometry-based proteomics. It provides tools for processing, analyzing, and interpreting proteomics data generated by liquid chromatography-mass spectrometry (LC-MS) experiments.

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Lab products found in correlation

Spectromine software, by Biognosys (2 mentions) Proteome discoverer software, by Thermo Fisher Scientific (1 mentions) Spss statistics ver 22, by IBM (1 mentions)

9 protocols using spectronaut pulsar x software

1

DIA Protein Identification and Quantification

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Raw data of DDA were searched against the National Center for Biotechnology Information database (1492129-1492124-Homo_sapiens. fasta; 130184 sequences) using Spectronaut Pulsar X software (Biognosys AG, Schlieren, Switzerland). Parameters for the searches were as follows: mass tolerance for precursor ion, 10 ppm; mass tolerance for product ion, 0.02 Da; maximum of missed cleavage sites, 2; fixed modification, carbamidomethyl; dynamic modification, oxidation of methionine; N-terminal modification, acetylation. Retrieval results were further filtered by Spectronaut Pulsar X software (Biognosys AG) to select identified peptide spectrum matches (PSMs) with the confidence level greater than 99%. Identified PSMs were then subjected to the verification of false discovery rate (FDR) for the removal of peptides and proteins with FDR greater than 1%. Then the DDA spectral library was established.
DIA data was imported into Spectronaut Pulsar X software (Biognosys AG). By search DDA spectral library, qualitative and quantitative analysis of peptides were implemented. Parameters set for DIA analysis were as follows: retention time correction, dynamic iRT; precursor ion Q value cutoff, 0.01.
Wang Z., Huang X., Tan H., Liang J., Li Z, & Shen J. (2023). Proteomic Comparison of Paraspinal Muscle Imbalance Between Idiopathic Scoliosis and Congenital Scoliosis. Neurospine, 20(2), 709-724.
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2

DIA-based Proteomic Analysis of Tbc1d10c T Cells

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Tbc1d10c+/+ and Tbc1d10c−/− splenic CD8 T cells (n = 3 replicates per group), were subjected to Data Independent Acquisition (DIA)-based proteomic analysis47 (link) by the Proteomics and Macromolecular Crystallography Shared Resource Core at Columbia University. Peptides were prepared as previously described48 (link) and injected on Orbitrap Fusion™ Tribrid™ mass spectrometer MS/MS analysis. DIA data were analyzed with directDIA 2.0 (Deep learning augmented spectrum-centric DIA analysis) in Spectronaut Pulsar X software (Biognosys). The default settings were used for targeted analysis of DIA data in Spectronaut, and DIA files were searched against the mouse UniProt fasta database. Results obtained from Spectronaut were further analyzed using the Spectronaut statistical package. Significantly changed protein abundance was determined by an unpaired t-test with a threshold for significance of p < .05 (permutation-based FDR correction) and 0.58 log2FC. Data sets can be accessed via the MassIVE repository (MSV000090556).
Cohen A.O., Woo S.H., Zhang J., Cho J., Ruiz M.E., Gong J., Du R., Yarygina O., Jafri D.Z., Bachelor M.A., Finlayson M.O., Soni R.K., Hayden M.S, & Owens D.M. (2022). Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response. Oncoimmunology, 11(1), 2141011.
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3

Proteome Discoverer-based Protein Identification

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The DDA data of 10 fractions were processed using Proteome Discoverer software (version 2.1, Thermo Scientific) and searched against the Swiss-Prot Human database (released in 2018, including 20,346 sequences) appended with the iRT peptide sequence. The search parameters were set as follows: two missed trypsin cleavage sites were allowed; the parent ion mass tolerances were set to 10 ppm; the fragment ion mass tolerances were set to 0.02 Da; the carbamidomethyl of cysteine was set as a fixed modification; and the oxidation of methionine was set as a variable modification. The false discovery rate (FDR) of proteins was less than 1%. A total of 2,184 protein groups, 11,518 peptide groups and 59,341 peptide spectrum matches were identified. The search result was used to set the variable windows for DIA mode. For the generation of spectral library, the DDA raw files were imported to Spectronaut Pulsar X software (Biognosys, Switzerland). All DIA raw files were processed using Spectronaut Plusar X software with default setting. All results were filtered by a Q value cutoff of 0.01. The protein identification was based on two unique peptides.
Meng W., Huan Y, & Gao Y. (2021). Urinary proteome profiling for children with autism using data-independent acquisition proteomics. Translational Pediatrics, 10(7), 1765-1778.
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4

Quantitative Proteomics of Klotho Treatment

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Plasma samples were prepared and analysed by data-independent acquisition liquid chromatography–mass spectrometry at Biognosys using a 2 h segmented gradient as previously described54 (link). For spectral library generation, 12 high-pH reversed phase chromatography fractions were analyzed by data-dependent acquisition liquid chromatography–mass spectrometry and searched against a mouse protein database (Uniprot without isoforms, 2018-07-01) using SpectroMine software (Biognosys). Data-independent acquisition data were analyzed with Spectronaut Pulsar X software (Biognosys), and data were filtered for a detection false discovery rate <1% on peptide and protein level. Peptide intensities were normalized using local regression normalization as implemented in Spectronaut. On average 505 protein groups (14,341 peptide precursors) were quantified in each run, and 535 protein groups (18,447 peptide precursors) were quantified at least once across the samples. Statistical analysis between vehicle- and klotho-treated samples was performed in Spectronaut using default settings and controlled for using Benjamini–Hochberg correction.
Park C., Hahn O., Gupta S., Moreno A.J., Marino F., Kedir B., Wang D., Villeda S.A., Wyss-Coray T, & Dubal D.B. (2023). Platelet factors are induced by longevity factor klotho and enhance cognition in young and aging mice. Nature Aging, 3(9), 1067-1078.
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5

Targeted Analysis of DIA Data

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DIA data were analyzed with directDIA 2.0, a spectral library-free analysis pipeline featured in Spectronaut Pulsar X software (Biognosys AG). The default settings were used for targeted analysis of DIA data in Spectronaut except the decoy generation was set to ‘mutated’. False discovery rate (FDR) was estimated using the mProphet approach (Reiter et al., 2011 (link)) and set to 1% at peptide precursor level and at 1% at protein level. For peptides and proteins that were not detected in a given sample (directDIA output as ‘Filtered’), the intensity was set to 0 for downstream analysis.
Hobson B.D., Choi S.J., Mosharov E.V., Soni R.K., Sulzer D, & Sims P.A. (2022). Subcellular proteomics of dopamine neurons in the mouse brain. eLife, 11, e70921.
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6

Quantitative Proteomics of Klotho Treatment

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Plasma samples were prepared and analysed by data-independent acquisition liquid chromatography–mass spectrometry at Biognosys using a 2 h segmented gradient as previously described54 (link). For spectral library generation, 12 high-pH reversed phase chromatography fractions were analyzed by data-dependent acquisition liquid chromatography–mass spectrometry and searched against a mouse protein database (Uniprot without isoforms, 2018-07-01) using SpectroMine software (Biognosys). Data-independent acquisition data were analyzed with Spectronaut Pulsar X software (Biognosys), and data were filtered for a detection false discovery rate <1% on peptide and protein level. Peptide intensities were normalized using local regression normalization as implemented in Spectronaut. On average 505 protein groups (14,341 peptide precursors) were quantified in each run, and 535 protein groups (18,447 peptide precursors) were quantified at least once across the samples. Statistical analysis between vehicle- and klotho-treated samples was performed in Spectronaut using default settings and controlled for using Benjamini–Hochberg correction.
Park C., Hahn O., Gupta S., Moreno A.J., Marino F., Kedir B., Wang D., Villeda S.A., Wyss-Coray T, & Dubal D.B. (2023). Platelet factors are induced by longevity factor klotho and enhance cognition in young and aging mice. Nature Aging, 3(9), 1067-1078.
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7

Comprehensive Protein Expression Analysis

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Raw data from data-dependent acquisition were processed and analyzed by Spectronaut Pulsar X software (Biognosys AG), which was set up to search the Uniprot_HomoSapiens_20367_20200226 database (http://www.uniprot.org; accessed on 6 may 2022), assuming that trypsin was the digestion enzyme. Carbamidomethyl (C) was specified as the fixed modification. Acetyl (protein N-term) and oxidation (M) were specified as the variable modifications. The FDR cutoff for the precursor and peptide levels was 1%. Spectronaut Pulsar X software was also applied for DIA analysis with default settings, and the FDR was also set as 1%.
A histogram of the frequency distribution of peptide length was used to evaluate whether the protease used in the study was reasonable. To intuitively compare the differences in protein expression levels between the two groups, a histogram of the frequency distribution of the ratios of the proteins found in each group (experimental vs. control) was used. Compared with the control group, proteins with FC ≤ 0.667 or ≥1.5 (p < 0.05) were considered to be DEPs. Correlation coefficient matrix creation, volcano plot construction, hierarchical clustering, heatmap construction, and statistical analyses were all conducted in R software.
Li H., Xu Y., Zhou X., Jin T., Wang Z., Sun Y., Wang H., Jiang D., Yin C., Shen B, & Song K. (2022). DIA-Based Proteomic Analysis of Plasma Protein Profiles in Patients with Severe Acute Pancreatitis. Molecules, 27(12), 3880.
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8

Proteomic Analysis of Clinical Data

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Clinical data were analyzed using IBM SPSS Statistics ver. 22.0 (IBM Co., Armonk, NY, USA). Continuous variables were presented as mean±standard deviation and compared by independent t-test or Mann-Whitney U-test. Categorical variables were presented as percentages (%) and compared by chi-square test. Proteomic data were processed by Spectronaut Pulsar X software (Biognosys AG) with default settings, and the FDR was also set as 1%. Proteomic analysis between groups were compared by independent t-test. A p-value of < 0.05 was considered statistically significant. Hypergeometric test was applied for enrichment analyses of GO terms, KEGG pathways and IPR domains, with a p-value of < 0.05 was considered significant.
Wang Z., Huang X., Tan H., Liang J., Li Z, & Shen J. (2023). Proteomic Comparison of Paraspinal Muscle Imbalance Between Idiopathic Scoliosis and Congenital Scoliosis. Neurospine, 20(2), 709-724.
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9

DIA Data Analysis with directDIA 2.0

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DIA data were analyzed with directDIA 2.0, a spectral library-free analysis pipeline featured in Spectronaut Pulsar X software (Biognosys AG). The default settings were used for targeted analysis of DIA data in Spectronaut except the decoy generation was set to "mutated". False discovery rate (FDR) was estimated using the mProphet approach (Reiter et al., 2011) (link) and set to 1% at peptide precursor level and at 1% at protein level. For peptides and proteins that were not detected in a given sample (directDIA output as 'Filtered'), the intensity was set to 0 for downstream analysis.
Hobson B.D., Choi S.J., Soni R.K., Sulzer D., & Sims P.A. (2021). Subcellular proteomics of dopamine neurons in the mouse brain reveals axonal enrichment of proteins encoded by Parkinson’s disease-linked genes.
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