5 research outputs found
Automated sample preparation with SP3 for lowâinput clinical proteomics
Abstract Highâthroughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples from a variety of sources, including freshâfrozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented singleâpot solidâphaseâenhanced sample preparation (SP3) on a liquid handling robot for automated processing (autoSP3) of tissue lysates in a 96âwell format. AutoSP3 performs unbiased protein purification and digestion, and delivers peptides that can be directly analyzed by LCMS, thereby significantly reducing handsâon time, reducing variability in protein quantification, and improving longitudinal reproducibility. We demonstrate the distinguishing ability of autoSP3 to process lowâinput samples, reproducibly quantifying 500â1,000 proteins from 100 to 1,000 cells. Furthermore, we applied this approach to a cohort of clinical FFPE pulmonary adenocarcinoma (ADC) samples and recapitulated their separation into known histological growth patterns. Finally, we integrated autoSP3 with AFA ultrasonication for the automated endâtoâend sample preparation and LCMS analysis of 96 intact tissue samples. Collectively, this constitutes a generic, scalable, and costâeffective workflow with minimal manual intervention, enabling reproducible tissue proteomics in a broad range of clinical and nonâclinical applications
Proteaseâresistant streptavidin for interaction proteomics
Abstract Streptavidinâmediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidinâderived peptides impede protein identification by mass spectrometry. Here, we present an approach to chemically modify streptavidin, thus rendering it resistant to proteolysis by trypsin and LysC. This modification results in over 100âfold reduction of streptavidin contamination and in better coverage of proteins interacting with various biotinylated bait molecules (DNA, protein, and lipid) in an overall simplified workflow
IFN-Îł Drives human monocyte differentiation into highly proinflammatory macrophages that resemble a phenotype relevant to psoriasis
As key cells of the immune system, macrophages coordinate the activation and regulation of the immune response. Macrophages present a complex phenotype that can vary from homeostatic, proinflammatory, and profibrotic to anti-inflammatory phenotypes. The factors that drive the differentiation from monocyte to macrophage largely define the resultant phenotype, as has been shown by the differences found in M-CSF- and GM-CSF-derived macrophages. We explored alternative inflammatory mediators that could be used for in vitro differentiation of human monocytes into macrophages. IFN-g is a potent inflammatory mediator produced by lymphocytes in disease and infections. We used IFN-g to differentiate human monocytes into macrophages and characterized the cells at a functional and proteomic level. IFN-g alone was sufficient to generate macrophages (IFN-g Mf) that were phagocytic and responsive to polarization. We demonstrate that IFN-g Mf are potent activators of T lymphocytes that produce IL-17 and IFN-g. We identified potential markers (GBP-1, IP-10, IL-12p70, and IL-23) of IFN-g Mf and demonstrate that these markers are enriched in the skin of patients with inflamed psoriasis. Collectively, we show that IFN-g can drive human monocyte to macrophage differentiation, leading to bona fide macrophages with inflammatory characteristics
Functional States in Tumor-Initiating Cell Differentiation in Human Colorectal Cancer
Intra-tumor heterogeneity of tumor-initiating cell (TIC) activity drives colorectal cancer (CRC) progression and therapy resistance. Here, we used single-cell RNA-sequencing of patient-derived CRC models to decipher distinct cell subpopulations based on their transcriptional profiles. Cell type-specific expression modules of stem-like, transit amplifying-like, and differentiated CRC cells resemble differentiation states of normal intestinal epithelial cells. Strikingly, identified subpopulations differ in proliferative activity and metabolic state. In summary, we here show at single-cell resolution that transcriptional heterogeneity identifies functional states during TIC differentiation. Furthermore, identified expression signatures are linked to patient prognosis. Targeting transcriptional states associated to cancer cell differentiation might unravel novel vulnerabilities in human CRC