Identification and Characterization of Adipose Tissue-Derived Human Antibodies With "Anti-self" Specificity.

We have previously shown that the human obese adipose tissue (AT) contributes to increased secretion of adipocyte-specific IgG antibodies in individuals with obesity. This occurs without any exogenous stimulation, because the ongoing process of cell death in the obese AT leads to the release of "self" antigens able to induce chronic stimulation of B cells. We have identified several mechanisms responsible for the release of "self" antigens, such as hypoxia, cell cytotoxicity, and DNA damage. In this paper, we confirm and extend our initial observation on a different cohort of individuals, and we show that also the plasma of these individuals is enriched in IgG antibodies with specificities for adipocyte-derived antigens. Adipocyte-specific IgG secreted in the obese AT are significantly correlated with those present in plasma. Using immunoprecipitation and mass spectrometry, we have identified these antigenic specificities. The antigens are almost exclusively intracellular or cell-associated, usually not recognized as "self" antigens, but they are released by cells dying in the AT. We also show for the first time that the adipocytes in the obese AT contribute to the secretion of IgG autoimmune antibodies and this seems to be due to their expression of the antigen-presenting molecules CD1d and, to a much lesser extent, MHC class II, as our mechanistic experiments performed in mice have shown. These results may lead to the development of novel therapeutic strategies to control autoimmunity.

Quantitative Metabolomics Using Isotope Residue Outlier Analysis (IROA®) with Internal Standards.

Various research strategies involving biomarker discovery and mechanistic studies in system biology depend on reproducible and reliable quantification of all metabolites from tissue(s) of interest. Contemporary analytical methods rely on mass spectrometry-based targeted and/or untargeted metabolomics platforms. The robustness of these analyses depends on the cleanliness of the samples, accuracy of the database, resolution of the instrument, and, the most variable of the list, the personal preferences of the researcher and the instrument operator. In this chapter, we introduce a simple method to prepare murine liver samples and carry it through the Isotope Ratio Outlier Analysis (IROA®) pipeline. This pipeline encompasses sample preparation, LC-MS-based peak acquisition, proprietary software-based library creation, normalization, and quantification of metabolites. IROA® offers a unique platform to create and normalize a local library and account for run-to-run variability over years of acquisition using the internal standards (IROA®-IS) and long-term reference standards (IROA®-LTRS).

Immature and Mature Collagen Crosslinks Quantification Using High-Performance Liquid Chromatography and High-Resolution Mass Spectrometry in Orbitrap™.

Different methodologies for collagen quantification have been described in the past. Introduction of mass spectrometry combined with high-performance liquid chromatography (HPLC) is a high-resolution tool, which has generated novel applications in biomedical research. In this study, HPLC coupled to electrospray ionization (ESI) tandem mass spectrometry (HPLC-ESI-MS/MS) was used to characterize tissue samples from AVFs done in rats. These findings helped create a protocol for identifying and quantifying components of immature and mature collagen crosslink moieties. Two different internal standards were used: epinephrine and pyridoxine. Quantification curves were drawn by means of these standards. The goal of the experiment was to achieve accurate quantification with the minimum amount of sample. Time and cost of experiment were considerably minimized. Up to date, this method has not been tested for crosslinking quantification.

Utility of Moderate and High-Resolution Mass Spectrometry for Class-Specific Lipid Identification and Quantification.

The study of lipidomics has been dramatically enhanced by using mass spectrometry techniques.With the purpose of identification and/or quantification of the lipids object of study, mass spectrometry has been proven to be the best approach, where three main techniques are currently being used: electrospray ionization (ESI), liquid chromatography (LC/MS), and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (Han et al. Mass Spectrom Rev 31:134-178, 2012). In order to avoid the problems generated by gradient concentration using liquid chromatography, here we describe the method used for the lipidomics analysis using moderate and high-resolution mass spectrometry for class-specific lipid identification and quantification for phospholipid species using ESI-MS/MS.