Mass spectrometry-based draft of the mouse proteome.

The laboratory mouse ranks among the most important experimental systems for biomedical research and molecular reference maps of such models are essential informational tools. Here, we present a quantitative draft of the mouse proteome and phosphoproteome constructed from 41 healthy tissues and several lines of analyses exemplify which insights can be gleaned from the data. For instance, tissue- and cell-type resolved profiles provide protein evidence for the expression of 17,000 genes, thousands of isoforms and 50,000 phosphorylation sites in vivo. Proteogenomic comparison of mouse, human and Arabidopsis reveal common and distinct mechanisms of gene expression regulation and, despite many similarities, numerous differentially abundant orthologs that likely serve species-specific functions. We leverage the mouse proteome by integrating phenotypic drug (n > 400) and radiation response data with the proteomes of 66 pancreatic ductal adenocarcinoma (PDAC) cell lines to reveal molecular markers for sensitivity and resistance. This unique atlas complements other molecular resources for the mouse and can be explored online via ProteomicsDB and PACiFIC.

Prosit: proteome-wide prediction of peptide tandem mass spectra by deep learning.

In mass-spectrometry-based proteomics, the identification and quantification of peptides and proteins heavily rely on sequence database searching or spectral library matching. The lack of accurate predictive models for fragment ion intensities impairs the realization of the full potential of these approaches. Here, we extended the ProteomeTools synthetic peptide library to 550,000 tryptic peptides and 21 million high-quality tandem mass spectra. We trained a deep neural network, termed Prosit, resulting in chromatographic retention time and fragment ion intensity predictions that exceed the quality of the experimental data. Integrating Prosit into database search pipelines led to more identifications at >10× lower false discovery rates. We show the general applicability of Prosit by predicting spectra for proteases other than trypsin, generating spectral libraries for data-independent acquisition and improving the analysis of metaproteomes. Prosit is integrated into ProteomicsDB, allowing search result re-scoring and custom spectral library generation for any organism on the basis of peptide sequence alone.

Lactobacillus supports Clostridiales to restrict gut colonization by multidrug-resistant Enterobacteriaceae.

Infections by multidrug-resistant Enterobacteriaceae (MRE) are life-threatening to patients. The intestinal microbiome protects against MRE colonization, but antibiotics cause collateral damage to commensals and open the way to colonization and subsequent infection. Despite the significance of this problem, the specific commensals and mechanisms that restrict MRE colonization remain largely unknown. Here, by performing a multi-omic prospective study of hospitalized patients combined with mice experiments, we find that Lactobacillus is key, though not sufficient, to restrict MRE gut colonization. Lactobacillus rhamnosus and murinus increase the levels of Clostridiales bacteria, which induces a hostile environment for MRE growth through increased butyrate levels and reduced nutrient sources. This mechanism of colonization resistance, an interaction between Lactobacillus spp. and Clostridiales involving cooperation between microbiota members, is conserved in mice and patients. These results stress the importance of exploiting microbiome interactions for developing effective probiotics that prevent infections in hospitalized patients.

Selective multi-kinase inhibition sensitizes mesenchymal pancreatic cancer to immune checkpoint blockade by remodeling the tumor microenvironment.

KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) is highly immunosuppressive and resistant to targeted and immunotherapies. Among the different PDAC subtypes, basal-like mesenchymal PDAC, which is driven by allelic imbalance, increased gene dosage and subsequent high expression levels of oncogenic KRAS, shows the most aggressive phenotype and strongest therapy resistance. In the present study, we performed a systematic high-throughput combination drug screen and identified a synergistic interaction between the MEK inhibitor trametinib and the multi-kinase inhibitor nintedanib, which targets KRAS-directed oncogenic signaling in mesenchymal PDAC. This combination treatment induces cell-cycle arrest and cell death, and initiates a context-dependent remodeling of the immunosuppressive cancer cell secretome. Using a combination of single-cell RNA-sequencing, CRISPR screens and immunophenotyping, we show that this combination therapy promotes intratumor infiltration of cytotoxic and effector T cells, which sensitizes mesenchymal PDAC to PD-L1 immune checkpoint inhibition. Overall, our results open new avenues to target this aggressive and therapy-refractory mesenchymal PDAC subtype.

Challenges in Clinical Metaproteomics Highlighted by the Analysis of Acute Leukemia Patients with Gut Colonization by Multidrug-Resistant Enterobacteriaceae.

The microbiome has a strong impact on human health and disease and is, therefore, increasingly studied in a clinical context. Metaproteomics is also attracting considerable attention, and such data can be efficiently generated today owing to improvements in mass spectrometry-based proteomics. As we will discuss in this study, there are still major challenges notably in data analysis that need to be overcome. Here, we analyzed 212 fecal samples from 56 hospitalized acute leukemia patients with multidrug-resistant Enterobactericeae (MRE) gut colonization using metagenomics and metaproteomics. This is one of the largest clinical metaproteomic studies to date, and the first metaproteomic study addressing the gut microbiome in MRE colonized acute leukemia patients. Based on this substantial data set, we discuss major current limitations in clinical metaproteomic data analysis to provide guidance to researchers in the field. Notably, the results show that public metagenome databases are incomplete and that sample-specific metagenomes improve results. Furthermore, biological variation is tremendous which challenges clinical study designs and argues that longitudinal measurements of individual patients are a valuable future addition to the analysis of patient cohorts.