Multiplexed Dilute-and-Shoot Liquid Chromatography-Multiple-Reaction Monitoring Mass Spectrometry Clinical Assay for Metanephrines and Catecholamines in Human Urine.

Quantifying urinary catecholamines and metanephrines is essential for the clinical screening and diagnosis of neuroendocrine tumours. HPLC with electrochemical detection (HPLC-ECD) is commonly used for this type of analysis but requires extensive sample cleanup. Simple and rapid dilute-and-shoot LC-multiple-reaction monitoring (MRM)-MS assays have been developed for quantitating these analytes in urine but have not yet been validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.

Characterization of 53 Multiplexed Targeted Proteomics Assays for Verification Studies in Cancer Cell Lines.

The National Cancer Institute's Clinical Proteomics Tumor Analysis Consortium (CPTAC) was established to address the need for improved design, standardization, and validation of proteomics assays to enable better translation of biomarkers from the analytical lab to the clinic. Here, we applied CPTAC guidelines to characterize quantitative mass spectrometry (MS) assays in a new multiple reaction monitoring (MRM) proteomics panel. The panel of 50 proteins was developed in response to a previous study that identified a proteomic profile of altered translational control associated with response to a new cancer drug.

Iron Regulatory Protein 2 (IRP2) Deficiency Is Protective Against Resistive Breathing-induced Pulmonary Inflammation.

Iron regulatory protein 2 (IRP2), a post-transcriptional regulator of cellular iron metabolism has been associated with susceptibility to chronic obstructive pulmonary disease (COPD). Resistive breathing (RB) is the hallmark of the pathophysiology of obstructive airway diseases, especially during exacerbations, where increased mechanical stress is imposed on the lung. We have previously shown that RB, through tracheal banding, mimicking severe airway obstruction, induces pulmonary inflammation and injury in previously healthy mice.

Clinical Proteomics Reveals Vulnerabilities in Noninvasive Breast Ductal Carcinoma and Drives Personalized Treatment Strategies.

This study provides real-world evidence for DCIS, a disease for which currently no molecular tools or biomarkers exist, and gives an unbiased, comprehensive, and deep proteomic profile, identifying >380 actionable targets.

Reproducible protein quantitation of 270 human proteins at increased depth using nanoparticle-based fractionation and multiple reaction monitoring mass spectrometry with stable isotope-labelled internal standards.

Here we show that when using a mix of 274 light synthetic peptide standards (NAT) as surrogates for 270 human plasma proteins, as well as stable isotope-labelled standards (SIS) as normalizers (both from MRM Proteomics Inc.) for targeted quantitative analysis by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS), the Seer Proteograph™ platform allowed for the enrichment and absolute quantitation of up to an additional 62 targets (median) compared to two standard proteomic workflows without enrichment, representing an increase of 44%. The nanoparticle-based fractionation workflow resulted in improved reproducibility compared to a traditional proteomic workflow with no fractionation (median 8.

The Society for Immunotherapy of Cancer Perspective on Tissue-Based Technologies for Immuno-Oncology Biomarker Discovery and Application.

With immuno-oncology becoming the standard of care for a variety of cancers, identifying biomarkers that reliably classify patient response, resistance, or toxicity becomes the next critical barrier towards improving care. Multi-parametric, multi-omics, and computational platforms generating an unprecedented depth of data are poised to usher in the discovery of increasingly robust biomarkers for enhanced patient selection and personalized treatment approaches. Deciding which developing technologies to implement in clinical settings ultimately, applied either alone or in combination, relies on weighing pros and cons, from minimizing patient sampling to maximizing data outputs, and assessing reproducibility and representativeness of findings, while lessening data fragmentation towards harmonization.

Digitalomics - digital transformation leading to omics insights.

Introduction: Biomarker discovery is increasingly moving from single omics to multiomics, as well as from multi-cell omics to single-cell omics. These transitions have increasingly adopted digital transformation technologies to accelerate the progression from data to insight. Here, we will discuss the concept of 'digitalomics' and how digital transformation directly impacts biomarker discovery.

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.

An effective Caenorhabditis elegans CRISPR training module for high school and undergraduate summer research experiences in molecular biology.

Engaging in research experiences as a high school or undergraduate student interested in science, technology, engineering, and mathematics (STEM) is pivotal for their academic and professional development. A structured teaching framework can help cultivate a student's curiosity and passion for learning and research. In this study, an eight-week training program was created to encompass fundamental molecular biology principles and hands-on laboratory activities.

Circadian Control of the Response of Macrophages to Plasmodium Spp.-Infected Red Blood Cells.

Malaria is a serious vector-borne disease characterized by periodic episodes of high fever and strong immune responses that are coordinated with the daily synchronized parasite replication cycle inside RBCs. As immune cells harbor an autonomous circadian clock that controls various aspects of the immune response, we sought to determine whether the intensity of the immune response to Plasmodium spp., the parasite causing malaria, depends on time of infection.

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).

The acid sphingomyelinase inhibitor imipramine enhances the release of UV photoproduct-containing DNA in small extracellular vesicles in UVB-irradiated human skin.

Nucleic acids, lipids, and other cell components can be found within different types of extracellular vesicles (EVs), which include apoptotic bodies (ABs), large extracellular vesicles (LEVs), and small extracellular vesicles (SEVs). Release of LEVs from cells can be reduced by genetic or pharmacological inhibition of the enzyme acid sphinogomyelinase (aSMase), and indeed several studies have demonstrated a role for the clinically approved aSMase inhibitor imipramine in blocking LEV release, including in response to UVB exposure. Given that exposure of keratinocytes to UVB radiation results in the generation of UVR photoproducts in DNA that can subsequently be found in association with ABs and SEVs, we examined how imipramine impacts the release of extracellular DNA containing UVR photoproducts at an early time point after UVR exposure.

A Simplified Proteomics LC-MRM-MS Assay for Determination of apoE Genotypes in Plasma Samples.

Apolipoprotein E (apoE), a polymorphic plasma protein, plays a pivotal role in lipid transportation. The human apoE gene possesses three major alleles (ε2, ε3, and ε4), which differ by single amino acid (cysteine to arginine) substitutions. The ε4 allele represents the primary genetic risk factor for Alzheimer's disease (AD), whereas the ε2 allele protects against the disease.

Measurement of CYP1A2 and CYP3A4 activity by a simplified Geneva cocktail approach in a cohort of free-living individuals: a pilot study.

The cytochrome P450 enzyme subfamilies, including CYP3A4 and CYP1A2, have a major role in metabolism of a range of drugs including several anti-cancer treatments. Many factors including environmental exposures, diet, diseaserelated systemic inflammation and certain genetic polymorphisms can impact the activity level of these enzymes. As a result, the net activity of each enzyme subfamily can vary widely between individuals and in the same individual over time.

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.

Multiple reaction monitoring assays for large-scale quantitation of proteins from 20 mouse organs and tissues.

Mouse is the mammalian model of choice to study human health and disease due to its size, ease of breeding and the natural occurrence of conditions mimicking human pathology. Here we design and validate multiple reaction monitoring mass spectrometry (MRM-MS) assays for quantitation of 2118 unique proteins in 20 murine tissues and organs. We provide open access to technical aspects of these assays to enable their implementation in other laboratories, and demonstrate their suitability for proteomic profiling in mice by measuring normal protein abundances in tissues from three mouse strains: C57BL/6NCrl, NOD/SCID, and BALB/cAnNCrl.

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.

Sequential multi-omics analysis identifies clinical phenotypes and predictive biomarkers for long COVID.

The post-acute sequelae of COVID-19 (PASC), also known as long COVID, is often associated with debilitating symptoms and adverse multisystem consequences. We obtain plasma samples from 117 individuals during and 6 months following their acute phase of infection to comprehensively profile and assess changes in cytokines, proteome, and metabolome. Network analysis reveals sustained inflammatory response, platelet degranulation, and cellular activation during convalescence accompanied by dysregulation in arginine biosynthesis, methionine metabolism, taurine metabolism, and tricarboxylic acid (TCA) cycle processes.