Dataset Information

Towards comprehensive plasma proteomics by orthogonal protease digestion

ABSTRACT: Rapid and consistent protein identification across large clinical cohorts is an important goal for clinical proteomics. With the development of data-independent technologies (DIA/SWATH-MS), it is now possible to analyze hundreds of samples with great reproducibility and quantitative accuracy. However, this technology benefits from empirically derived spectral libraries that define the detectable set of peptides and proteins. Here we apply a simple and accessible tip-based workflow for the generation of spectral libraries to provide a comprehensive overview on the plasma proteome in individuals with and without active tuberculosis (TB). To boost protein coverage, we utilized non-conventional proteases such as GluC and AspN together with the gold standard trypsin, identifying more than 30,000 peptides mapping to 3,309 proteins. Application of this library to quantify plasma proteome differences in TB infection recovered more than 400 proteins in 50 minutes of MS-acquisition, including diagnostic Mycobacterium Tuberculosis(Mtb) proteins that have previously been detectable primarily by antibody-based assays and intracellular proteins not previously described to be in plasma.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human) Mycobacterium Tuberculosis Tkk-01-0032

TISSUE(S): Blood Plasma

DISEASE(S): Pulmonary Tuberculosis

SUBMITTER: Andrea Fossati

LAB HEAD: Danielle Swaney

PROVIDER: PXD025671 | Pride | 2021-07-29

REPOSITORIES: Pride

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Publications

Toward Comprehensive Plasma Proteomics by Orthogonal Protease Digestion.

Fossati Andrea A Richards Alicia L AL Chen Kuei-Ho KH Jaganath Devan D Cattamanchi Adithya A Ernst Joel D JD Swaney Danielle L DL

Journal of proteome research 20210728 8

Rapid and consistent protein identification across large clinical cohorts is an important goal for clinical proteomics. With the development of data-independent technologies (DIA/SWATH-MS), it is now possible to analyze hundreds of samples with great reproducibility and quantitative accuracy. However, this technology benefits from empirically derived spectral libraries that define the detectable set of peptides and proteins. Here, we apply a simple and accessible tip-based workflow for the gener ...[more]

PMID: 34319755

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Project description:The anabolic androgen 17M-NM-2-trenbolone (TB) can cause masculinization and reduce fecundity of fish. However, the underlying mechanisms of various biological pathways including metabolism, biosynthesis etc. are largely unknown. Here, we evaluated the effects of TB using the medaka DNA microarray representing 36,398 genes. Larval medaka, Oryzias latipes, (within 24 hrs posthatch) were exposed to TB at various concentrations (2, 6, 20, 60, 100, 200 ng/L) for up to 7 days. Dose-response relationships in gene expression levels of the categorized genes were analyzed using the cumulative chisquared method. Microarray analyses of the TB-exposed larvae showed that 117 and 32 genes were determined as up and down-regulated genes, respectively. The most significant GO term for biological process identified within this gene list was lipid metabolic process, which contained 26 genes in up-regulated genes. In this category, M-bM-^@M-^\cholesterol biosynthetic processM-bM-^@M-^] was highlighted as an important subcategory with 15 genes, including hydroxymethylglutaryl-CoA synthase cytoplasmic, squalene monooxygenase, lanosterol synthase etc. RT-PCR measurements in these genes were consistent with the microarray results in the direction and magnitude of these changes in gene expression. On the other hands, in the category of M-bM-^@M-^\sexual differentiation and developmentM-bM-^@M-^], genes related to hypothalamic-pituitary-gonadal (HPG) axis were not affected by TB treatment except for one gene encoded to cytochrome P450 19A1. Genes related to oogenesis, such as choriogenins and vitellogenins were weakly down regulated at 2-200 ng/L of TB. Our findings demonstrate that genes encoding cholesterol synthesis pathway via the mevalonate pathway were controlled by TB in larval medaka. TB concentrations used were 0 (control), 2, 6, 20, 60, 100 and 200 ng/L for 7 days of exposure. Each TB treatment had 90 larval medaka for each chamber. At day 7 of the exposures, triplicate samples (30 larvae/sample) from each chamber respectively were collected, flash-frozen in liquid nitrogen, and stored in liquid nitrogen until RNA extraction.

Dataset Information

Towards comprehensive plasma proteomics by orthogonal protease digestion

Publications

Toward Comprehensive Plasma Proteomics by Orthogonal Protease Digestion.

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