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Mass spectrometry-based proteomics and metabolomics are fast growing and powerful technologies, with the potential to revolutionize health care and precision medicine. Human blood plasma and serum are already the most established samples for clinical analysis and are analyzed today with antibody-based assays. However, immunoassays have some inherent limitations that could be overcome by MS-based proteomics, which should be the optimal technology to investigate changes in the human plasma proteome in a specific and unbiased manner. Recently, we developed an automated, rapid and robust shotgun proteomics workflow that allows us to analyse hundreds of plasma proteins. We call the workflow ‘plasma proteome profiling’(Geyer et al., Cell Syst., 2016) and we have already applied it to several clinical studies, comprising up to 1,300 plasma proteomes (Geyer et al., Mol. Syst. Biol., 2016). However, until now, available separation technology has severely limited throughput and robustness and thereby prevented proteomics (and metabolomics) technologies from being fully integrated and routinely used in a clinical setting.

The bacterial type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR Associated (Cas) systems, and in particular Streptococcus pyogenes CRISPR–Cas9, have been broadly applied to edit the genome of bacterial and eukaryotic cells. Cas9, which is an RNA-guided programmable nuclease, is a powerful tool for disrupting protein-coding genes. Cas9 cleaves target sites to generate a double-strand break (DSB) that is repaired via an error-prone repair process, leading to insertion/deletion …

The heart is a central human organ and its diseases are the leading cause of death worldwide, but an in-depth knowledge of the identity and quantity of its constituent proteins is still lacking. Here, we determine the healthy human heart proteome by measuring 16 anatomical regions and three major cardiac cell types by high-resolution mass spectrometry-based proteomics. From low microgram sample amounts, we quantify over 10,700 proteins in this high dynamic range tissue. We combine copy numbers per cell with protein organellar assignments to build a model of the heart proteome at the subcellular level. Analysis of cardiac fibroblasts identifies cellular receptors as potential cell surface markers. Application of our heart map to atrial fibrillation reveals individually distinct mitochondrial dysfunctions. The heart map is available at maxqb. biochem. mpg. de as a resource for future analyses of normal heart function …

Clinical analysis of blood is the most widespread diagnostic procedure in medicine, and blood biomarkers are used to categorize patients and to support treatment decisions. However, existing biomarkers are far from comprehensive and often lack specificity and new ones are being developed at a very slow rate. As described in this review, mass spectrometry (MS)‐based proteomics has become a powerful technology in biological research and it is now poised to allow the characterization of the plasma proteome in great depth. Previous “triangular strategies” aimed at discovering single biomarker candidates in small cohorts, followed by classical immunoassays in much larger validation cohorts. We propose a “rectangular” plasma proteome profiling strategy, in which the proteome patterns of large cohorts are correlated with their phenotypes in health and disease. Translating such concepts into clinical practice …

The interconnected PI3K and MAPK signaling pathways are commonly perturbed in cancer. Dual inhibition of these pathways by the small-molecule PI3K inhibitor pictilisib (GDC-0941) and the MEK inhibitor cobimetinib (GDC-0973) suppresses cell proliferation and induces cell death better than either single agent in several preclinical models. Using mass spectrometry-based phosphoproteomics, we have identified the RING finger E3 ubiquitin ligase RNF157 as a target at the intersection of PI3K and MAPK signaling. We demonstrate that RNF157 phosphorylation downstream of the PI3K and MAPK pathways influences the ubiquitination and stability of RNF157 during the cell cycle in an anaphase-promoting complex/cyclosome–CDH1-dependent manner. Deletion of these phosphorylation-targeted residues on RNF157 disrupts binding to CDH1 and protects RNF157 from ubiquitination and degradation …

Evolution of tumor cell phenotypes promotes heterogeneity and therapy resistance. Here we found that induction of CD73, the enzyme that generates immunosuppressive adenosine, is linked to melanoma phenotype switching. Activating MAPK mutations and growth factors drove CD73 expression, which marked both nascent and full activation of a mesenchymal-like melanoma cell state program. Proinflammatory cytokines like TNFα cooperated with MAPK signaling through the c-Jun/AP-1 transcription factor complex to activate CD73 transcription by binding to an intronic enhancer. In a mouse model of T-cell immunotherapy, CD73 was induced in relapse melanomas, which acquired a mesenchymal-like phenotype. We also detected CD73 upregulation in melanoma patients progressing under adoptive T-cell transfer or immune checkpoint blockade, arguing for an adaptive resistance mechanism. Our work …

Detection of cytosolic DNA constitutes a central event in the context of numerous infectious and sterile inflammatory conditions. Recent studies have uncovered a bipartite mode of cytosolic DNA recognition, in which the cGAS-STING axis triggers antiviral immunity, whereas AIM2 triggers inflammasome activation. Here, we show that AIM2 is dispensable for DNA-mediated inflammasome activation in human myeloid cells. Instead, detection of cytosolic DNA by the cGAS-STING axis induces a cell death program initiating potassium efflux upstream of NLRP3. Forward genetics identified regulators of lysosomal trafficking to modulate this cell death program, and subsequent studies revealed that activated STING traffics to the lysosome, where it triggers membrane permeabilization and thus lysosomal cell death (LCD). Importantly, the cGAS-STING-NLRP3 pathway constitutes the default inflammasome response during …

Regulatory T cells (Tregs) prevent autoimmunity but limit antitumor immunity. The canonical NF-κB signaling pathway both activates immunity and promotes thymic Treg development. Here, we report that mature Tregs continue to require NF-κB signaling through IκB-kinase β (IKKβ) after thymic egress. Mice lacking IKKβ in mature Tregs developed scurfy-like immunopathology due to death of peripheral FoxP3+ Tregs. Also, pharmacological IKKβ inhibition reduced Treg numbers in the circulation by ∼50% and downregulated FoxP3 and CD25 expression and STAT5 phosphorylation. In contrast, activated cytotoxic T lymphocytes (CTLs) were resistant to IKKβ inhibition because other pathways, in particular nuclear factor of activated T cells (NFATc1) signaling, sustained their survival and expansion. In a melanoma mouse model, IKKβ inhibition after CTL cross-priming improved the antitumor response and delayed …

Purpose: Human exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports different cargo proteins out of the nucleus. Inducing nuclear accumulation of these proteins by inhibiting XPO1 causes cancer cell death. First clinical validation of pharmacological inhibition of XPO1 was obtained with the Selective Inhibitor of Nuclear Export (SINE) compound selinexor (KPT-330) demonstrating activity in phase-II/IIb clinical trials when dosed 1 to 3 times weekly. The second-generation SINE compound KPT-8602 shows improved tolerability and can be dosed daily. Here, we investigate and validate the drug–target interaction of KPT-8602 and explore its activity against acute lymphoblastic leukemia (ALL). Experimental Design: We examined the effect of KPT-8602 on XPO1 function and XPO1-cargo as well as on a panel of leukemia cell lines. Mutant XPO1 leukemia cells were …

Glioblastoma multiformes (GBMs) are high-grade astrocytomas and the most common brain malignancies. Primary GBMs are often associated with disturbed RAS signaling, and expression of oncogenic HRAS results in a malignant phenotype in glioma cell lines. Secondary GBMs arise from lower-grade astrocytomas, have slower progression than primary tumors, and contain IDH1 mutations in over 70% of cases. Despite significant amount of accumulating genomic and transcriptomic data, the fundamental mechanistic differences of gliomagenesis in these two types of high-grade astrocytoma remain poorly understood. Only a few studies have attempted to investigate the proteome, phosphorylation signaling, and epigenetic regulation in astrocytoma. In the present study, we applied quantitative phosphoproteomics to identify the main signaling differences between oncogenic HRAS and mutant IDH1-driven glioma …

Recent advances in mass spectrometry (MS)-based proteomics now allow very deep coverage of cellular proteomes. To achieve near-comprehensive identification and quantification, the combination of a first HPLC-based peptide fractionation orthogonal to the on-line LC-MS/MS step has proven to be particularly powerful. This first dimension is typically performed with milliliter/min flow and relatively large column inner diameters, which allow efficient pre-fractionation but typically require peptide amounts in the milligram range. Here, we describe a novel approach termed “spider fractionator” in which the post-column flow of a nanobore chromatography system enters an eight-port flow-selector rotor valve. The valve switches the flow into different flow channels at constant time intervals, such as every 90 s. Each flow channel collects the fractions into autosampler vials of the LC-MS/MS system. Employing a freely …

Protein glycation is a concentration-dependent nonenzymatic reaction of reducing sugars with amine groups of proteins to form early as well as advanced glycation (end-) products (AGEs). Glycation is a highly disease-relevant modification but is typically only studied on a few blood proteins. To complement our blood proteomics studies in diabetics, we here investigate protein glycation by higher energy collisional dissociation (HCD) fragmentation on Orbitrap mass spectrometers. We established parameters to most efficiently fragment and identify early glycation products on in vitro glycated model proteins. Retaining standard collision energies does not degrade performance if the most dominant neutral loss of H6O3 is included into the database search strategy. Glycation analysis of the entire HeLa proteome revealed an unexpected intracellular preponderance for arginine over lysine modification in early and …