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Evaluation of iTRAQ and SWATH-MS for the Quantification of Proteins Associated with Insulin Resistance in Human Duodenal Biopsy Samples.

ABSTRACT: Insulin resistance (IR) is associated with increased production of triglyceride-rich lipoproteins of intestinal origin. In order to assess whether insulin resistance affects the proteins involved in lipid metabolism, we used two mass spectrometry based quantitative proteomics techniques to compare the intestinal proteome of 14 IR patients to that of 15 insulin sensitive (IS) control patients matched for age and waist circumference. A total of 3886 proteins were identified by the iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry approach and 2290 by the SWATH-MS strategy (Serial Window Acquisition of Theoretical Spectra). Using these two methods, 208 common proteins were identified with a confidence corresponding to FDR < 1%, and quantified with p-value < 0.05. The quantification of those 208 proteins has a Pearson correlation coefficient (r2) of 0.728 across the two techniques. Gene Ontology analyses of the differentially expressed proteins revealed that annotations related to lipid metabolic process and oxidation reduction process are overly represented in the set of under-expressed proteins in IR subjects. Furthermore, both methods quantified proteins of relevance to IR. These data also showed that SWATH-MS is a promising and compelling alternative to iTRAQ for protein quantitation of complex mixtures.

SUBMITTER: Bourassa S

PROVIDER: S-EPMC4423961 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Evaluation of iTRAQ and SWATH-MS for the quantification of proteins associated with insulin resistance in human duodenal biopsy samples

Project description:Insulin resistance (IR) is associated with increased production of triglyceride-rich lipoproteins of intestinal origin. In order to assess whether insulin resistance affects the proteins involved in lipid metabolism, we used two mass spectrometry based quantitative proteomics techniques to compare the intestinal proteome of 14 IR patients to that of 16 insulin sensitive (IS) control patients matched for age and waist circumference. A total of 3896 proteins were identified by the iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry approach and 2311 by the SWATH-MS strategy (Serial Window Acquisition of Theoretical Spectra). Using these two methods, 208 common proteins were identified with a confidence corresponding to FDR < 1%, and quantified with p-value < 0.05. The quantification of those 208 proteins has a Pearson correlation coefficient (r2) of 0.728 across the two techniques. Gene Ontology analyses of the differentially expressed proteins revealed that annotations related to lipid metabolic process and oxidation reduction process are overly represented in the set of under-expressed proteins in IR subjects. Furthermore, both methods quantified proteins of relevance to IR. These data also showed that SWATH-MS is a promising and compelling alternative to iTRAQ for protein quantitation of complex mixtures.

2015-08-26 | PXD001506 | Pride

Evaluation of iTRAQ and SWATH-MS for the quantification of proteins associated with insulin resistance in human duodenal biopsy samples

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A Prostate Cancer Proteomics Database for SWATH-MS Based Protein Quantification.

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Project description:Consistent detection and quantification of protein post-translational modifications (PTMs) across sample cohorts is a prerequisite for functional analysis of biological processes. Data-independent acquisition (DIA) is a bottom-up mass spectrometry approach that provides complete information on precursor and fragment ions. However, owing to the convoluted structure of DIA data sets, confident, systematic identification and quantification of peptidoforms has remained challenging. Here, we present inference of peptidoforms (IPF), a fully automated algorithm that uses spectral libraries to query, validate and quantify peptidoforms in DIA data sets. The method was developed on data acquired by the DIA method SWATH-MS and benchmarked using a synthetic phosphopeptide reference data set and phosphopeptide-enriched samples. IPF reduced false site-localization by more than sevenfold compared with previous approaches, while recovering 85.4% of the true signals. Using IPF, we quantified peptidoforms in DIA data acquired from >200 samples of blood plasma of a human twin cohort and assessed the contribution of heritable, environmental and longitudinal effects on their PTMs.

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Corneal proteome and differentially expressed corneal proteins in highly myopic chicks using a label-free SWATH-MS quantification approach

Project description:Myopia, or short-sightedness, is a highly prevalent refractive disorder in which the eye’s focal length is too short for its axial dimension in its relaxed state. High myopia is associated with increased risks of blinding ocular complications and abnormal eye shape. In addition to consistent findings on posterior segment anomalies in high myopia (e.g., scleral remodeling), more recent biometric and biomechanical data in myopic humans and animal models also indicate anterior segment anomalies (e.g., corneal biomechanical properties). Because the cornea is the anterior-most ocular tissue, providing essential refractive power and physiological stability, it is important to understand the biochemical signaling pathway during myopia development. This study first aimed to establish the entire chicken corneal proteome. Then, using the classical form deprivation paradigm to induce high myopia in chicks, state-of-the-art bioinformatics technologies were applied to identify eight differentially expressed proteins in the highly myopic cornea. These results provide strong foundation for future corneal research, especially those using chicken as an animal model for myopia development.

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Probing SWATH-MS as a tool for proteome level quantification in a nonmodel fish.

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Quantification of Dynamic Protein Interactions and Phosphorylation in LPS Signaling Pathway by SWATH-MS.

Project description:Lipopolysaccharide (LPS)-induced macrophage activation is a prototype of innate immune response. Although key effector proteins in LPS signaling pathway have been revealed, the map of dynamic protein interactions and phosphorylation as well as the stoichiometry of protein complexes are lacking. Here we present a dynamic map of protein interactions and phosphorylation in MyD88, TRAF6 and NEMO complexes obtained by SWATH-MS. The comprehensive MS measurement leads to quantification of up to about 3,000 proteins across about 21-40 IP samples. We detected and quantified almost all known interactors of MyD88, TRAF6 and NEMO. By analyzing these quantitative data, we uncovered differential recruitment of IRAK family proteins to LPS-induced signaling complexes and determined the stoichiometry of the Myddosome complex. In addition, quantitative phosphoproteomics analysis identified a number of unreported high-confidence phosphosites on the key proteins in LPS signaling pathway. Collectively, data of dynamic protein interactions and phosphorylation presented by this study could be a resource for further study of the LPS signaling pathway.

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