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Numerous studies have proven the potential of cytokeratin 19 fragment 21-1 (CYFRA 21-1) detection in the (early) diagnosis and treatment monitoring of non-small cell lung cancer (NSCLC). Conventional immunoassays for CYFRA 21-1 quantification are however prone to interferences and lack diagnostic sensitivity and standardization. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach based on a different, often superior, detection principle, which may improve the clinical applicability of CYFRA 21-1 in cancer diagnostics. Therefore, we developed and validated a protein precipitation, immunoaffinity (IA) LC-MS/MS assay for quantitative analysis of serum CYFRA 21-1. Selective sample preparation was performed using ammonium sulfate (AS) precipitation, IA purification, tryptic digestion and LC-MS/MS quantification using a signature peptide and isotopically labeled internal standard. The workflow was optimized and validated according to EMA guidelines and results were compared to a conventional immunoassay. Significant interference effects were seen during IA purification, which were sufficiently solved by performing AS precipitation prior to IA purification. A linear calibration curve was obtained in the range of 1.0-100 » ng/mL (R2=0.98). Accuracy and precision were well within acceptance criteria. In sera of patients suspected of lung cancer, the method showed good correlation with the immunoassay. A robust AS precipitation-IA LC-MS/MS assay for the quantification of serum CYFRA 21-1 was developed. With this assay, the clinically added value of LC-MS/MS-based detection over immunoassays can be further explored.

OBJECTIVES: Timely diagnosis of lung cancer (LC) is crucial to achieve optimal patient care and outcome. Moreover, the number of procedures required to obtain a definitive diagnosis can have a large influence on the life expectancy of a patient. Here, adherence with existing Dutch guidelines for timeliness and type and number of invasive and imaging procedures was assessed.MATERIALS AND METHODS: 1096 patients with suspected LC were enrolled in this multicenter prospective study (NL9146). The overall survival, time from referral to the first appointment with the pulmonologist, time to diagnosis and treatment, and the number of imaging and invasive procedures were evaluated. Patients were divided into different diagnostic groupsearly- and advanced stage non-small-cell lung cancer (NSCLC), small-cell lung cancer (SCLC), large cell neuroendocrine carcinoma of the lung (LCNEC), patients without LC and patients without a definitive diagnosis.RESULTS: The majority of patients (66 %) received a definitive diagnosis within 5 weeks, although the time to diagnosis of early-stage LC patients and patients without LC was significantly longer comparted to advanced stage LC. An increase in invasive procedures was seen for early-stage LC compared to advanced stage LC and for 13 % of the advanced stage non-squamous NSCLC patients up to three additional invasive procedures were performed solely to obtain sufficient material for NGS. For patients without a definitive diagnosis, 50 % did undergo at least one invasive procedure, while 11 % did not wish to undergo any invasive procedures.CONCLUSION: These insights could aid in improved LC diagnostics and efficient implementation of new techniques like liquid biopsy and artificial intelligence. This may lead to more timely LC care, a decreased number of invasive procedures, less variability between the diagnostic trajectory of different patients and aid in obtaining a definitive diagnosis for all patients.

SRC homology 3 (SH3) domains are critical interaction modules that orchestrate the assembly of protein complexes involved in diverse biological processes. They facilitate transient protein–protein interactions by selectively interacting with proline-rich motifs (PRMs). A database search revealed 298 SH3 domains in 221 human proteins. Multiple sequence alignment of human SH3 domains is useful for phylogenetic analysis and determination of their selectivity towards PRM-containing peptides (PRPs). However, a more precise functional classification of SH3 domains is achieved by constructing a phylogenetic tree only from PRM-binding residues and using existing SH3 domain–PRP structures and biochemical data to determine the specificity within each of the 10 families for particular PRPs. In addition, the C-terminal proline-rich domain of the RAS activator SOS1 covers 13 of the 14 recognized proline-rich consensus sequence motifs, encompassing differential PRP pattern selectivity among all SH3 families. To evaluate the binding capabilities and affinities, we conducted fluorescence dot blot and polarization experiments using 25 representative SH3 domains and various PRPs derived from SOS1. Our analysis has identified 45 interacting pairs, with binding affinities ranging from 0.2 to 125 micromolar, out of 300 tested and potential new SH3 domain-SOS1 interactions. Furthermore, it establishes a framework to bridge the gap between SH3 and PRP interactions and provides predictive insights into the potential interactions of SH3 domains with PRMs based on sequence specifications. This novel framework has the potential to enhance the understanding of protein networks mediated by SH3 domain–PRM interactions and be utilized as a general approach for other domain–peptide interactions.

Membraneless organelles are important for spatial organization of proteins and regulation of intracellular processes. Proteins can be recruited to these condensates by specific protein–protein or protein–nucleic acid interactions, which are often regulated by post-translational modifications. However, the mechanisms behind these dynamic, affinity-based protein recruitment events are not well understood. Here, a coacervate system that incorporates the 14-3-3 scaffold protein to study enzymatically regulated recruitment of 14-3-3-binding proteins is presented, which mostly bind in a phosphorylation-dependent manner. Synthetic coacervates are efficiently loaded with 14-3-3, and phosphorylated binding partners, such as the c-Raf pS233/pS259 peptide (c-Raf), show 14-3-3-dependent sequestration with up to 161-fold increase in local concentration. The c-Raf domain is fused to green fluorescent protein (GFP-c-Raf) to demonstrate recruitment of proteins. In situ phosphorylation of GFP-c-Raf by a kinase leads to enzymatically regulated uptake. The introduction of a phosphatase into coacervates preloaded with the phosphorylated 14-3-3-GFP-c-Raf complex results in a significant cargo efflux mediated by dephosphorylation. Finally, the general applicability of this platform to study protein–protein interactions is demonstrated by the phosphorylation-dependent and 14-3-3-mediated active reconstitution of a split-luciferase inside artificial cells. This work presents an approach to study dynamically regulated protein recruitment in condensates, using native interaction domains.

Molecular glues are powerful tools for the control of protein-protein interactions. Yet, the mechanisms underlying multi-component protein complex formation remain poorly understood. Native mass spectrometry (MS) detects multiple protein species simultaneously, providing an entry to elucidate these mechanisms. Here, for the first time, covalent molecular glue stabilization was kinetically investigated by combining native MS with biophysical and structural techniques. This approach elucidated the stoichiometry of a multi-component protein-ligand complex, the assembly order, and the contributions of covalent versus non-covalent binding events that govern molecular glue activity. Aldehyde-based molecular glue activity is initially regulated by cooperative non-covalent binding, followed by slow covalent ligation, further enhancing stabilization. This study provides a framework to investigate the mechanisms of covalent small molecule ligation and informs (covalent) molecular glue development.

Mathematical modelling of molecular systems can be extremely helpful in elucidating complex phenomena in (bio)chemistry. However, equilibrium conditions in systems consisting of more than two components, such as for molecular glues bound to two proteins, can typically not be analytically determined without assumptions and (semi-)numerical models are not trivial to derive by the non-expert. Here we present a framework for equilibrium models, geared towards molecular glues and other contemporary multicomponent chemical biology challenges. The framework utilizes a general derivation method capable of generating custom mass-balance models for equilibrium conditions of complex molecular systems, based on the simple, reversible biomolecular reactions describing these systems. Several chemical biology concepts are revisited via the framework to demonstrate the simplicity, generality and validity of the approach. The ease of use of the framework and the ability to both analyze systems and gain additional insights in the underlying parameters driving equilibria formation strongly aids the analysis and understanding of biomolecular systems. New directions for research and analysis are brought forward based on the model formation and system and parameter analysis. This conceptual framework severely reduces the time and expertise requirements which currently impede the broad integration of such valuable equilibrium models into molecular glue development and chemical biology research.