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.

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.

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.

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.