No abstract

Keloid disease is characterized by hyperproliferation of responsive fibroblasts with vigorously continuous synthesis of extracellular matrix (ECM) components. Although the process by which keloids develop is poorly understood, most theories of the etiology are referred to fibroblast dysfunction. A central event in dermal repair is the release of growth factors in response to skin injury, which leads to the dysregulation of several crucial pathways that initiate the activation of keloid fibroblasts (KFs) and promote ECM accumulation. Hence, strategies aimed at reducing the production of these cytokines and/or disrupting their intracellular signal transduction have potential clinical significance for curing keloid. As the first oral multikinase inhibitor, sorafenib blocks a number of intracellular signaling pathways which are also pivotal for keloid pathogenesis. Therefore, evaluation of the effects of sorafenib on keloid disease seems timely and pertinent. In this study, we reported the identification of sorafenib that antagonized TGF-β/Smad and MAPK/ERK signaling pathways in primary KFs. Impressively, treatment with sorafenib inhibited KF cell proliferation, migration, and invasion, and simultaneously reduced collagen production in KFs. Furthermore, we present ex vivo evidence that sorafenib induced the arrest of KF migration, the inhibition of angiogenesis, and the reduction of collagen accumulation. These preclinical observations suggest that sorafenib deserves systematic exploration as a candidate agent for the future treatment of keloids.Key message The intracellular TGF-β/Smad and MAPK/ERK signaling pathways is blocked by sorafenib.Sorafenib inhibits the proliferation, migration, invasion, and ECM deposition in keloid fibroblasts.Sorafenib reduces KF migration and concomitantly angiogenesis in keloid explants.Sorafenib is a promising agent for the treatment of keloids and hypertrophic scars. Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-016-1430-3) contains supplementary material, which is available to authorized users.

For quantitative proteomics, efficient, robust, and reproducible sample preparation with high throughput is critical yet challenging, especially when large cohorts are involved, as is often required by clinical/pharmaceutical studies. We describe a rapid and straightforward surfactant cocktail-aided extraction/precipitation/on-pellet digestion (SEPOD) strategy to address this need. Prior to organic solvent precipitation and on-pellet digestion, SEPOD treats samples with a surfactant cocktail (SC) containing multiple nonionic/anionic surfactants, which achieves (i) exhaustive/reproducible protein extraction, including membrane-bound proteins; (ii) effective removal of detrimental nonprotein matrix components (e.g., >94% of phospholipids); (iii) rapid/efficient proteolytic digestion owing to dual (surfactants + precipitation) denaturation. The optimal SC composition and concentrations were determined by Orthogonal-Array-Design investigation of their collective/individuals effects on protein extraction/denaturation. Key parameters for cleanup and digestion were experimentally identified as well. The optimized SEPOD procedures allowed a rapid 6 h digestion providing a clean digest with high peptide yields and excellent quantitative reproducibility (especially low-abundance proteins). Compared with filter-assisted sample preparation (FASP) and in-solution digestion, SEPOD showed superior performance by recovering substantially more peptide/proteins (including integral membrane proteins), yielding significantly higher peptide intensities and improving quantification for peptides with extreme physicochemical properties. SEPOD was further applied in a large-cohort temporal investigation of 44 IAV-infected mouse lungs, providing efficient and reproducible peptide yields (77.9 ± 4.6%) across all samples. With the IonStar pipeline, >6 400 unique protein groups were quantified (≥2 peptide/protein, peptide-FDR < 0.05%), ∼99% without missing data in any sample with <7% technical median-intragroup CV. Altered proteome patterns revealed interesting novel insights into pathophysiological changes by IAV infection. In summary, SEPOD offers a feasible solution for rapid, efficient, and reproducible preparation of biological samples, facilitating high-quality proteomic quantification of large sample cohorts.

In the last decade, the advancement of liquid chromatography mass spectrometry (LC/MS) techniques has enabled their broad application in protein characterization, both quantitatively and qualitatively. Owing to certain important merits of LC/MS techniques (e.g., high selectivity, flexibility, and rapid method development), LC/MS assays are often deemed as preferable alternatives to conventional methods (e.g., ligand-binding assays) for the analysis of protein biotherapeutics. At the discovery and development stages, LC/MS is generally employed for two purposes absolute quantification of protein biotherapeutics in biological samples and qualitative characterization of proteins. For absolute quantification of a target protein in bio-matrices, recent work has led to improvements in the efficiency of LC/MS method development, sample treatment, enrichment and digestion, and high-performance low-flow-LC separation. These advances have enhanced analytical sensitivity, specificity, and robustness. As to qualitative analysis, a range of techniques have been developed to characterize intramolecular disulfide bonds, glycosylation, charge variants, primary sequence heterogeneity, and the drug-to-antibody ratio of antibody drug conjugate (ADC), which has enabled a refined ability to assess product quality. In this review, we will focus on the discussion of technical challenges and strategies of LC/MS-based quantification and characterization of biotherapeutics, with the emphasis on the analysis of antibody-based biotherapeutics such as monoclonal antibodies (mAbs) and ADCs. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:734-754, 2017.