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. Upon cleavage of the covalently linked peptide drug, a chemical remnant of a defined mass remains on the bound amino acid, which is then used to unambiguously identify the drug binding site. Applying our approach to known peptide-drug/protein pairs with reported crystal structures, such as the calmodulin-melittin interaction, we were able to validate the identified binding sites based on structural models. Overall, our cleavable photoaffinity labeling strategy represents a powerful tool to enable the identification of protein targets and specific binding sites of a wide variety of bioactive peptides in the future.

Instrumentos para avaliação da sexualidade de mulheres grávidas: revisão integrativa / Instruments for assessing the sexuality of pregnant women: integrative review / Instrumentos para evaluar la sexualidad de mujeres embarazadas: revisión integrativa

Objetivo: Identificar instrumentos utilizados para avaliar aspectos inerentes à sexualidade de mulheres grávidas. Método: Revisão integrativa da literatura realizada de agosto a setembro de 2022. A busca ocorreu nas bases de dados LILACS, MEDLINE, BDENF, IBECS, CINAHL e na biblioteca SciELO. Foram utilizados os descritores Pregnancy; Surveys and Questionnaires; Quiz; Forms; Instruments; Sexuality; Sexual behavior; Sexual Dysfunction, Physiological; Sexual Function; eSexual Dysfunction.Resultados:Foram identificados 5.632 estudos. Após aplicação dos critérios de inclusão e exclusão e leitura dos textos na íntegra, a amostra foi composta por 27 artigos. Foram identificados 14 instrumentos. Predominou a utilização de questionários com destaque para o Female Sexual Function Index(FSFI) por vezes associado a outro instrumento de coleta de dados. As abordagens direcionaram-se prioritariamente à função sexual de mulheres grávidas e ocorreram em serviços de saúde direcionados ao atendimento desse público. Conclusões: Evidenciaram-se instrumentos aplicáveis para investigar aspectos inerentes à sexualidade de mulheres grávidas úteis para aplicação no contexto do cuidado clínico de enfermagem durante a assistência pré-natal, o que pode contribuir para a integralidade da atenção à saúde sexual.

Objective: To identify instruments used to assess aspects inherent to the sexuality of pregnantwomen. Method:Integrative literature review occurred from August to September 2022. LILACS, MEDLINE, BDENF, IBECS, CINAHL databases, and the SciELO library were the chosen databases for the search. The descriptors Pregnancy; Surveys and Questionnaires; Quiz; Forms; Instruments; Sexuality; Sexual behavior; Sexual Dysfunction, Physiological; Sexual Function; and Sexual Dysfunction. Results:5,632 studies were identified. After applying the inclusion and exclusion criteria and reading the texts in full, the sample consisted of 27 articles. We identified 14 instruments in the studies. The use of questionnaires predominated, with emphasis on the Female Sexual Function Index (FSFI) associated with another tool for data collection. The approaches were directed primarily to the sexual function of pregnant women and occurred in health services assisting this public. Conclusions:Applicable instruments investigate aspects inherent to the sexuality of pregnant women, useful for application in the context of clinical nursing care during prenatal care, which can contribute to the comprehensiveness of sexual health care.

Objetivo: Identificar instrumentos utilizados para evaluar aspectos inherentes a la sexualidad de las mujeres embarazadas. Método:Revisión integrativa de la literatura realizada de agosto a septiembre de 2022. Las bases de datos LILACS, MEDLINE, BDENF, IBECS, CINAHL y la biblioteca SciELO fueron las bases de datos elegidas para la búsqueda. Los descriptores Embarazo; Encuestas y Cuestionarios; Prueba; formularios; instrumentos; Sexualidad; Comportamiento sexual; Disfunción Sexual Fisiológica; función sexual; y disfunción sexual. Resultados:Se identificaron 5.632 estudios. Después de aplicar los criterios de inclusión y exclusión y leer los textos en su totalidad, la muestra quedó constituida por 27 artículos. Se identificaron 14 instrumentos en los estudios. Predominó el uso de cuestionarios, con énfasis en el Índice de Función Sexual Femenina (FSFI)asociado a otra herramienta de recolección de datos. Los abordajes estaban dirigidos principalmente a la función sexual de las mujeres embarazadas y ocurrieron en los servicios de salud que atendían a ese público. Conclusiones:Los instrumentos aplicablesinvestigan aspectos inherentes a la sexualidad de la gestante, útiles para su aplicación en el contexto de la atención clínica de enfermería durante el prenatal, que pueden contribuir a la integralidad de la atención a la salud sexual.

Utility, promise, and limitations of liquid chromatography-mass spectrometry-based therapeutic drug monitoring in precision medicine.

Therapeutic drug monitoring (TDM) is typically referred to as the measurement of the concentration of drugs in patient blood. Although in the past, TDM was restricted to drugs with a narrow therapeutic range in order to avoid drug toxicity, TDM has recently become a major tool for precision medicine being applied to many more drugs. Through compensating for interindividual differences in a drug's pharmacokinetics, improved dosing of individual patients based on TDM ensures maximum drug effectiveness while minimizing side effects. This is especially relevant for individuals that present a particularly high intervariability in pharmacokinetics, such as newborns, or for critically/severely ill patients. In this article, we will review the applications for and limitations of TDM, discuss for which patients TDM is most beneficial and why, examine which techniques are being used for TDM, and demonstrate how mass spectrometry is increasingly becoming a reliable and convenient alternative for the TDM of different classes of drugs. We will also highlight the advances, challenges, and limitations of the existing repertoire of TDM methods and discuss future opportunities for TDM-based precision medicine.

Interactome Analysis of KIN (Kin17) Shows New Functions of This Protein.

KIN (Kin17) protein is overexpressed in a number of cancerous cell lines, and is therefore considered a possible cancer biomarker. It is a well-conserved protein across eukaryotes and is ubiquitously expressed in all cell types studied, suggesting an important role in the maintenance of basic cellular function which is yet to be well determined. Early studies on KIN suggested that this nuclear protein plays a role in cellular mechanisms such as DNA replication and/or repair; however, its association with chromatin depends on its methylation state. In order to provide a better understanding of the cellular role of this protein, we investigated its interactome by proximity-dependent biotin identification coupled to mass spectrometry (BioID-MS), used for identification of protein-protein interactions. Our analyses detected interaction with a novel set of proteins and reinforced previous observations linking KIN to factors involved in RNA processing, notably pre-mRNA splicing and ribosome biogenesis. However, little evidence supports that this protein is directly coupled to DNA replication and/or repair processes, as previously suggested. Furthermore, a novel interaction was observed with PRMT7 (protein arginine methyltransferase 7) and we demonstrated that KIN is modified by this enzyme. This interactome analysis indicates that KIN is associated with several cell metabolism functions, and shows for the first time an association with ribosome biogenesis, suggesting that KIN is likely a moonlight protein.

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.

Targeted and Untargeted Proteomics Approaches in Biomarker Development.

An enormous amount of research effort has been devoted to biomarker discovery and validation. With the completion of the human genome, proteomics is now playing an increasing role in this search for new and better biomarkers. Here, what leads to successful biomarker development is reviewed and how these features may be applied in the context of proteomic biomarker research is considered. The "fit-for-purpose" approach to biomarker development suggests that untargeted proteomic approaches may be better suited for early stages of biomarker discovery, while targeted approaches are preferred for validation and implementation. A systematic screening of published biomarker articles using MS-based proteomics reveals that while both targeted and untargeted technologies are used in proteomic biomarker development, most researchers do not combine these approaches. i) The reasons for this discrepancy, (ii) how proteomic technologies can overcome technical challenges that seem to limit their translation into the clinic, and (iii) how MS can improve, complement, or replace existing clinically important assays in the future are discussed.

The kin17 Protein in Murine Melanoma Cells.

kin17 has been described as a protein involved in the processes of DNA replication initiation, DNA recombination, and DNA repair. kin17 has been studied as a potential molecular marker of breast cancer. This work reports the detection and localization of this protein in the murine melanoma cell line B16F10-Nex2 and in two derived subclones with different metastatic potential, B16-8HR and B16-10CR. Nuclear and chromatin-associated protein fractions were analyzed, and kin17 was detected in all fractions, with an elevated concentration observed in the chromatin-associated fraction of the clone with low metastatic potential, suggesting that the kin17 expression level could be a marker of melanoma.

NOR sites detected by Ag-dAPI staining of an unusual autosome chromosome of Bradysia hygida (Diptera:Sciaridae) colocalize with C-banded heterochromatic region.

The study of chromosomes in insects is a good tool in mitotic process analysis, zoographic localization and evolution investigation. Among them, the Sciaridae offers a karyotype with a small number of chromosomes, where the heterochromatin and nucleolar organizer region, NOR, are easily analyzed in metaphase chromosomes obtained from cerebral ganglia squashes. In this work, the heterochromatic regions on Bradysia hygida mitotic chromosomes, revealed by C-banding, were identified as centromeric blocks on A and C chromosomes and as dark interstitial region in B and X chromosomes. By Ag-DAPI staining, active nucleolus organizer region, NOR, was revealed associated to the constitutive heterochromatin in the end of the C autosome chromosome. The C-band regions and the unusual ribosomal site localization are discussed.