Various factors, including drugs as well as non-molecular influences, induce alterations in the stability of proteins in cell lysates, living cells and organisms. These alterations can be probed by applying a stability-modifying agent, such as elevated temperature, to a varying degree. As a second dimension of variation, drug concentration or factor intensity can be used. However, the corresponding analysis scheme has a low throughput and high cost. Additionally, since traditional data analysis employs curve fitting, proteins with unusual behavior are frequently ignored. The novel Proteome Integral Stability Alteration (PISA) assay avoids these issues altogether, increasing the analysis throughput by one to two orders of magnitude for unlimited number of parameter variation points. The consumption of the compound and biological material decreases by the same factor. We envision widespread use of the PISA approach in chemical biology and drug development..

Blood-based anti-amyloid-β (Aβ) immunoglobulins (IgGs) and peripheral inflammation are factors correlating with development of Alzheimer's disease (AD). IgG functionality can drastically change from anti- to pro-inflammatory via alterations in the IgG-Fc N-glycan structure. Herein, we tested if IgG-Fc glycosylation in plasma is indeed altered during the development of AD. Samples from age-matched subjects of 23 controls, 58 patients with stable mild cognitive impairment (SMCI), 34 patients with progressive (P)MCI, and 31 patients with AD were investigated. Label-free shotgun proteomics was applied without glycoprotein enrichment. Glycans on peptides EEQYNSTYR (IgG1) and EEQFNSTFR (IgG2) were quantified, and their abundances were normalized to total IgGn glycoform abundance. Univariate and multivariate statistics were employed to investigate the correlations between the patients groups and the abundances of the IgG glycoforms as well as those of inflammatory mediating proteins. Significant differences (p ≤ 0.05) were found, with a lower abundance of complex galactosylated and sialylated forms in AD. For females, a decline in glycoform complexity correlated with disease progress but an inverse change was found in males prior to the onset of AD. Principal component analysis (PCA; Males: R(2)X(cum) = 0.65, Q(2)(cum) = 0.34; Females: R(2)X(cum) = 0.62, Q(2)(cum) = 0.36), confirmed the gender similarities (for controls, SMCI and AD) as well as differences (for PMCI), and showed a close correlation between pro-inflammatory protein markers, AD, female PMCI, and truncated IgG-Fc glycans. The differences observed between genders prior to the onset of AD may indicate a lower ability in females to suppress peripheral inflammation, which may lead to exacerbated disease progression.

The data show for the first time how ACPA, derived from human RA, recognize citrulline. The specific citrulline recognition and backbone-mediated interactions provide a structural explanation of the promiscuous recognition of citrullinated peptides by RA-specific ACPAs. This article is protected by copyright. All rights reserved.

Oxylipins (e.g. eicosanoids) are endogenous signaling molecules that are formed from fatty acids by mono- or dioxygenase-catalyzed oxygenation and have been shown to play an important role in pathophysiological processes in the lung. These lipid mediators have been extensively studied for their role in inflammation in a broad swathe of respiratory diseases including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis and extrinsic allergic alveolitis. Traditional efforts have employed analytical methods (e.g. radio- and enzyme-immunoassay techniques) capable of measuring a limited number of compounds simultaneously. The advent of the omics technologies is changing this approach and methods are being developed for the quantification of small molecules (i.e. metabolomics) as well as lipid-focused efforts (i.e. lipidomics). This review examines in detail the breadth of oxylipins and their biological activity in the respiratory system. In addition, the state-of-the-art methodology in profiling of oxylipins via mass spectrometry is summarized including sample work-up and data processing. These methods will greatly increase our ability to probe oxylipin biology and examine for cross-talk between biological pathways as well as specific compartments in the body. These new data will increase our insight into disease processes and have great potential to identify new biomarkers for disease diagnosis as well as novel therapeutic targets.

BackgroundAsthma is a respiratory tract disorder characterized by airway hyper-reactivity and chronic inflammation. Allergic asthma is associated with the production of allergen-specific IgE and expansion of allergen-specific T-cell populations. Progression of allergic inflammation is driven by T-helper type 2 (Th2) mediators and is associated with alterations in the levels of lipid mediators.ObjectivesResponses of the respiratory system to birch allergen provocation in allergic asthmatics were investigated. Eicosanoids and other oxylipins were quantified in the bronchoalveolar lumen to provide a measure of shifts in lipid mediators associated with allergen challenge in allergic asthmatics.MethodsEighty-seven lipid mediators representing the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP) metabolic pathways were screened via LC-MS/MS following off-line extraction of bronchoalveolar lavage fluid (BALF). Multivariate statistics using OPLS were employed to interrogate acquired oxylipin data in combination with immunological markers.ResultsThirty-two oxylipins were quantified, with baseline asthmatics possessing a different oxylipin profile relative to healthy individuals that became more distinct following allergen provocation. The most prominent differences included 15-LOX-derived ω-3 and ω-6 oxylipins. Shared-and-Unique-Structures (SUS)-plot modeling showed a correlation (R2 = 0.7) between OPLS models for baseline asthmatics (R2Y[cum] = 0.87, Q2[cum] = 0.51) and allergen-provoked asthmatics (R2Y[cum] = 0.95, Q2[cum] = 0.73), with the majority of quantified lipid mediators and cytokines contributing equally to both groups. Unique structures for allergen provocation included leukotrienes (LTB4 and 6-trans-LTB4), CYP-derivatives of linoleic acid (epoxides/diols), and IL-10.ConclusionsDifferences in asthmatic relative to healthy profiles suggest a role for 15-LOX products of both ω-6 and ω-3 origin in allergic inflammation. Prominent differences at baseline levels indicate that non-symptomatic asthmatics are subject to an underlying inflammatory condition not observed with other traditional mediators. Results suggest that oxylipin profiling may provide a sensitive means of characterizing low-level inflammation and that even individuals with mild disease display distinct phenotypic profiles, which may have clinical ramifications for disease.