Clinical proteomics reveals vulnerabilities in non-invasive breast ductal carcinoma and drives personalized treatment strategies.

Ductal carcinoma in situ (DCIS) is the most common type (80%) of non-invasive breast lesions in women. The lack of validated prognostic markers, limited patient numbers, and variable tissue quality have a significant impact on diagnosis, risk stratification, patient enrolment, and the results of clinical studies. Here, we performed label-free quantitative proteomics on 50 clinical formalin-fixed, paraffin embedded biopsies, validating 22 putative biomarkers from independent genetic studies.

Proteomic and cytokine profiling of a CTRP8-RXFP1 glioma mouse model.

Glioblastoma (GB) is the most prevalent and aggressive primary brain tumor with fatal outcome due to a lack of effective treatments. We previously identified C1q-tumor necrosis factor-related protein 8 (CTRP8), a new member of the adiponectin family, as a novel agonist of the relaxin family peptide receptor 1 (RXFP1) and showed that the CTRP8-RXFP1 ligand-receptor system facilitates increased invasiveness and chemoresistance in GB cells. In the present study, we have investigated the role of the CTRP8-RXFP1 signaling axis in glioma progression using an orthotopic mouse model xenografted with human U251 glioma cells stably expressing CTRP8 and RXFP1.

Blocking tumor-intrinsic MNK1 kinase restricts metabolic adaptation and diminishes liver metastasis.

Dysregulation of the mitogen-activated protein kinase interacting kinases 1/2 (MNK1/2)-eukaryotic initiation factor 4E (eIF4E) signaling axis promotes breast cancer progression. MNK1 is known to influence cancer stem cells (CSCs); self-renewing populations that support metastasis, recurrence, and chemotherapeutic resistance, making them a clinically relevant target. The precise function of MNK1 in regulating CSCs, however, remains unexplored.

Proteomic evidence of depression-associated astrocytic dysfunction in the human male olfactory bulb.

The olfactory bulb (OB), a major structure of the limbic system, has been understudied in human investigations of psychopathologies such as depression. To explore more directly the molecular features of the OB in depression, a global comparative proteome analysis was carried out with human post-mortem OB samples from 11 males having suffered from depression and 12 healthy controls. We identified 188 differentially abundant proteins (with adjusted p < 0.

HNRNPA1 de novo Variant Associated with Early Childhood Onset, Rapidly Progressive Generalized Myopathy.

HNRNPA1 variants are known to cause degenerative motoneuron and muscle diseases which manifests in middle age or later. We report on a girl with early childhood onset, rapidly progressive generalized myopathy including ultrastructural findings in line with a proteinopathy. Proteomics of patient-derived muscle and combined screening of genomic data for copy number variations identified a HNRNPA1 de novo intragenic deletion as causative for the phenotype.

Paradigm Shift: Major Role of Ion-Pairing-Dependent Size Exclusion Effects in Bottom-Up Proteomics Reversed-Phase Peptide Separations.

Can reversed-phase peptide retention be the same for C8 and C18 columns? or increase for otherwise identical columns with a smaller surface area? Can replacing trifluoroacetic acid (TFA) with formic acid (FA) improve the peak shape? According to our common understanding of peptide chromatography, absolutely not. Surprisingly, a thorough comparison of the peptide separation selectivity of 100 and 120 Å fully porous C18 sorbents to maximize the performance of our in-house proteomics LC-MS/MS setup revealed an unexpectedly higher peptide retentivity for a wider pore packing material, despite it having a smaller surface area. Concurrently, the observed increase in peptide retention─which drives variation in separation selectivity between 100 and 120 Å pore size materials─was more pronounced for smaller peptides.

Establishing Personalized Blood Protein Reference Ranges Using Noninvasive Microsampling and Targeted Proteomics: Implications for Antidoping Strategies.

To prevent doping practices in sports, the World Anti-Doping Agency implemented the Athlete Biological Passport (ABP) program, monitoring biological variables over time to indirectly reveal the effects of doping rather than detect the doping substance or the method itself. In the context of this program, a highly multiplexed mass spectrometry-based proteomics assay for 319 peptides corresponding to 250 proteins was developed, including proteins associated with blood-doping practices. "Baseline" expression profiles of these potential biomarkers in capillary blood (dried blood spots (DBS)) were established using multiple reaction monitoring (MRM).

Accelerating Proteomics Using Broad Specificity Proteases.

Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches. Thus, multienzyme strategies are used to maximize sequence coverage in post-translational modification profiling. We present fast and robust SP3- and STRAP-based protocols for the broad-specificity proteases subtilisin, proteinase K, and thermolysin.

The Deubiquitylase Otub1 Regulates the Chemotactic Response of Splenic B Cells by Modulating the Stability of the γ-Subunit Gng2.

The ubiquitin proteasome system performs the covalent attachment of lysine 48-linked polyubiquitin chains to substrate proteins, thereby targeting them for degradation, while deubiquitylating enzymes (DUBs) reverse this process. This posttranslational modification regulates key features both of innate and adaptative immunity, including antigen presentation, protein homeostasis and signal transduction. Here we show that loss of one of the most highly expressed DUBs, Otub1, results in changes in murine splenic B cell subsets, leading to a significant increase in marginal zone and transitional B cells and a concomitant decrease in follicular B cells.

Hyperinsulinemia acts via acinar insulin receptors to initiate pancreatic cancer by increasing digestive enzyme production and inflammation.

The rising pancreatic cancer incidence due to obesity and type 2 diabetes is closely tied to hyperinsulinemia, an independent cancer risk factor. Previous studies demonstrated reducing insulin production suppressed pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in Kras-mutant mice. However, the pathophysiological and molecular mechanisms remained unknown, and in particular it was unclear whether hyperinsulinemia affected PanIN precursor cells directly or indirectly.

Compendium of Chromatographic Behavior of Post-translationally and Chemically Modified Peptides in Bottom-Up Proteomic Experiments.

We have systematically evaluated the chromatographic behavior of post-translationally/chemically modified peptides using data spanning over 70 of the most relevant modifications. These retention properties were measured for standard bottom-up proteomic settings (fully porous C18 separation media, 0.1% formic acid as ion-pairing modifier) using collections of modified/nonmodified peptide pairs.

Improved Coverage of the N-Terminome by Combining ChaFRADIC with Alternative Proteases.

Many proteolytic cleavage events cannot be covered with conventional trypsin-based N-terminomics workflows. These typically involve the derivatization of protein N-termini and Lys residues as an initial step, such that trypsin will cleave C-terminal of arginine but not lysine residues (ArgC-like cleavage). From 20,422 reviewed human protein sequences in Uniprot, 3597 have known N-terminal signal peptides.

FGF1 supports glycolytic metabolism through the estrogen receptor in endocrine-resistant and obesity-associated breast cancer.

Background: Obesity increases breast cancer risk and breast cancer-specific mortality, particularly for people with estrogen receptor (ER)-positive tumors. Body mass index (BMI) is used to define obesity, but it may not be the best predictor of breast cancer risk or prognosis on an individual level. Adult weight gain is an independent indicator of breast cancer risk.

Mapping Peptide-Protein Interactions by Amine-Reactive Cleavable Photoaffinity Reagents.

Photoaffinity labeling followed by tandem mass spectrometry is an often used strategy to identify protein targets of small-molecule drugs or drug candidates, which, under ideal conditions, enables the identification of the actual drug binding site. In the case of bioactive peptides, however, identifying the distinct binding site is hampered because of complex fragmentation patterns during tandem mass spectrometry. We here report the development and use of small cleavable photoaffinity reagents that allow functionalization of bioactive peptides for light-induced covalent binding to their protein targets.

Omics approaches for the assessment of biological responses to nanoparticles.

Nanotechnology has enabled the development of innovative therapeutics, diagnostics, and drug delivery systems. Nanoparticles (NPs) can influence gene expression, protein synthesis, cell cycle, metabolism, and other subcellular processes. While conventional methods have limitations in characterizing responses to NPs, omics approaches can analyze complete sets of molecular entities that change upon exposure to NPs.

Exploring the variable space of shallow machine learning models for reversed-phase retention time prediction.

Peptide retention time (RT) prediction algorithms are tools to study and identify the physicochemical properties that drive the peptide-sorbent interaction. Traditional RT algorithms use multiple linear regression with manually curated parameters to determine the degree of direct contribution for each parameter and improvements to RT prediction accuracies relied on superior feature engineering. Deep learning led to a significant increase in RT prediction accuracy and automated feature engineering via chaining multiple learning modules.

Discovery-Based Proteomics Identify Skeletal Muscle Mitochondrial Alterations as an Early Metabolic Defect in a Mouse Model of β-Thalassemia.

Although metabolic complications are common in thalassemia patients, there is still an unmet need to better understand underlying mechanisms. We used unbiased global proteomics to reveal molecular differences between the th mouse model of thalassemia and wild-type control animals focusing on skeletal muscles at 8 weeks of age. Our data point toward a significantly impaired mitochondrial oxidative phosphorylation.

Offline Peptide Fractionation and Parallel Reaction Monitoring MS for the Quantitation of Low-Abundance Plasma Proteins.

Mass spectrometry (MS)-based protein quantitation is an attractive means for research and diagnostics due to its high specificity, precision, sensitivity, versatility, and the ability to develop multiplexed assays for the "absolute" quantitation of virtually any protein target. However, due to the large dynamic range of protein concentrations in blood, high abundance proteins in blood plasma hinder the detectability and quantification of lower-abundance proteins which are often relevant in the context of different diseases. Here we outline a streamlined method involving offline high-pH reversed-phase fractionation of human plasma samples followed by the quantitative analysis of specific fractions using nanoLC-parallel reaction monitoring (PRM) on a Q Exactive Plus mass spectrometer for peptide detection and quantitation with increased sensitivity.

Inhibition of the MNK1/2-eIF4E Axis Augments Palbociclib-Mediated Antitumor Activity in Melanoma and Breast Cancer.

Aberrant cell-cycle progression is characteristic of melanoma, and CDK4/6 inhibitors, such as palbociclib, are currently being tested for efficacy in this disease. Despite the promising nature of CDK4/6 inhibitors, their use as single agents in melanoma has shown limited clinical benefit. Herein, we discovered that treatment of tumor cells with palbociclib induces the phosphorylation of the mRNA translation initiation factor eIF4E.

Multi-omics approaches to study platelet mechanisms.

Platelets are small anucleate cell fragments (2-4 μm in diameter) in the blood, which play an essential role in thrombosis and hemostasis. Genetic or acquired platelet dysfunctions are linked to bleeding, increased risk of thromboembolic events and cardiovascular diseases. Advanced proteomic approaches may pave the way to a better understanding of the roles of platelets in hemostasis, and pathophysiological processes such as inflammation, metastatic spread and thrombosis.