Pressure overload induces ISG15 to facilitate adverse ventricular remodeling and promote heart failure.

Inflammation promotes adverse ventricular remodeling, a common antecedent of heart failure. Here, we set out to determine how inflammatory cells affect cardiomyocytes in the remodeling heart. Pathogenic cardiac macrophages induced an IFN response in cardiomyocytes, characterized by upregulation of the ubiquitin-like protein IFN-stimulated gene 15 (ISG15), which posttranslationally modifies its targets through a process termed ISGylation. Cardiac ISG15 is controlled by type I IFN signaling, and ISG15 or ISGylation is upregulated in mice with transverse aortic constriction or infused with angiotensin II; rats with uninephrectomy and DOCA-salt, or pulmonary artery banding; cardiomyocytes exposed to IFNs or CD4+ T cell-conditioned medium; and ventricular tissue of humans with nonischemic cardiomyopathy. By nanoscale liquid chromatography-tandem mass spectrometry, we identified the myofibrillar protein filamin-C as an ISGylation target. ISG15 deficiency preserved cardiac function in mice with transverse aortic constriction and led to improved recovery of mouse hearts ex vivo. Metabolomics revealed that ISG15 regulates cardiac amino acid metabolism, whereas ISG15 deficiency prevented misfolded filamin-C accumulation and induced cardiomyocyte autophagy. In sum, ISG15 upregulation is a feature of pathological ventricular remodeling, and protein ISGylation is an inflammation-induced posttranslational modification that may contribute to heart failure development by altering cardiomyocyte protein turnover.

Early Prediction of COVID-19 Patient Survival by Targeted Plasma Multi-Omics and Machine Learning.

The recent surge of coronavirus disease 2019 (COVID-19) hospitalizations severely challenges healthcare systems around the globe and has increased the demand for reliable tests predictive of disease severity and mortality. Using multiplexed targeted mass spectrometry assays on a robust triple quadrupole MS setup which is available in many clinical laboratories, we determined the precise concentrations of hundreds of proteins and metabolites in plasma from hospitalized COVID-19 patients. We observed a clear distinction between COVID-19 patients and controls and, strikingly, a significant difference between survivors and nonsurvivors. With increasing length of hospitalization, the survivors' samples showed a trend toward normal concentrations, indicating a potential sensitive readout of treatment success. Building a machine learning multi-omic model that considers the concentrations of 10 proteins and five metabolites, we could predict patient survival with 92% accuracy (area under the receiver operating characteristic curve: 0.97) on the day of hospitalization. Hence, our standardized assays represent a unique opportunity for the early stratification of hospitalized COVID-19 patients.

An Update on MRMAssayDB: A Comprehensive Resource for Targeted Proteomics Assays in the Community.

Precise multiplexed quantification of proteins in biological samples can be achieved by targeted proteomics using multiple or parallel reaction monitoring (MRM/PRM). Combined with internal standards, the method achieves very good repeatability and reproducibility enabling excellent protein quantification and allowing longitudinal and cohort studies. A laborious part of performing such experiments lies in the preparation steps dedicated to the development and validation of individual protein assays. Several public repositories host information on targeted proteomics assays, including NCI's Clinical Proteomic Tumor Analysis Consortium assay portals, PeptideAtlas SRM Experiment Library, SRMAtlas, PanoramaWeb, and PeptideTracker, with all offering varying levels of details. We introduced MRMAssayDB in 2018 as an integrated resource for targeted proteomics assays. The Web-based application maps and links the assays from the repositories, includes comprehensive up-to-date protein and sequence annotations, and provides multiple visualization options on the peptide and protein level. We have extended MRMAssayDB with more assays and extensive annotations. Currently it contains >828 000 assays covering >51 000 proteins from 94 organisms, of which >17 000 proteins are present in >2400 biological pathways, and >48 000 mapping to >21 000 Gene Ontology terms. This is an increase of about four times the number of assays since introduction. We have expanded annotations of interaction, biological pathways, and disease associations. A newly added visualization module for coupled molecular structural annotation browsing allows the user to interactively examine peptide sequence and any known PTMs and disease mutations, and map all to available protein 3D structures. Because of its integrative approach, MRMAssayDB enables a holistic view of suitable proteotypic peptides and commonly used transitions in empirical data. Availability: http://mrmassaydb.proteincentre.com.

Direct and Precise Measurement of Bevacizumab Levels in Human Plasma Based on Controlled Methionine Oxidation and Multiple Reaction Monitoring.

Bevacizumab is a monoclonal antibody which targets vascular endothelial growth factor A (VEGF-A) and is used to treat various cancers and recently COVID-19. The dosage recommendations for bevacizumab are determined on the basis of body weight, and the drug is administered after defined time intervals, when it is presumed to still be above its minimum effective serum concentration. Interindividual and disease-stage-related variations in bevacizumab catabolism, however, can affect the proper dosing of patients, resulting in plasma concentrations which may not be within the optimal therapeutic window for the drug. Therapeutic drug monitoring (TDM) enables the assessment of patients' serum concentrations and allows personalized dosing which has the potential to improve efficacy and reduce side effects. While TMD is often performed using ligand-based assays, mass spectrometry (MS)-based TDM offers improved specificity. Here, we present a robust multiple reaction monitoring (MRM)-MS-based TDM method for the precise quantification of bevacizumab plasma concentrations, based on the controlled oxidation of the methionine-containing peptide, STAYLQMNSLR. The assay shows good linearity (r 2 = 0.9951), robustness, and precision (CVs < 20%) for the quantification of bevacizumab, with a lower limit of quantification (S/N > 10) of 1.8 µg/mL of plasma, without the need for enrichment and requiring less than 1 µL of plasma and less than 6 h from sampling to result.

Mass-Spectrometric Detection of SARS-CoV-2 Virus in Scrapings of the Epithelium of the Nasopharynx of Infected Patients via Nucleocapsid N Protein.

The detection of viral RNA by polymerase chain reaction (PCR) is currently the main diagnostic tool for COVID-19 ( Eurosurveillance 2019, 25 (3), 1). The PCR-based test, however, shows limited sensitivity, especially in the early and late stages of disease development ( Nature 2020, 581, 465-469; J. Formosan Med. Assoc. 2020, 119 (6) 1123), and is relatively time-consuming. Fast and reliable complementary methods for detecting the viral infection would be of help in the current pandemic conditions. Mass spectrometry is one of such possibilities. We have developed a mass-spectrometry-based method for the detection of the SARS CoV-2 virus in nasopharynx epithelial swabs based on the detection of the viral nucleocapsid N protein. Our approach shows confident identification of the N protein in patient samples, even those with the lowest viral loads, and a much simpler preparation procedure. Our main protocol consists of virus inactivation by heating and the addition of isopropanol and tryptic digestion of the proteins sedimented from the swabs followed by MS analysis. A set of unique peptides, produced as a result of proteolysis of the nucleocapsid phosphoprotein of SARS-CoV-2, is detected. The obtained results can further be used to create fast parallel mass-spectrometric approaches for the detection of the virus in the nasopharyngeal mucosa, saliva, sputum and other physiological fluids.