Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. Areas covered: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. Expert commentary: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine.

Exosomes are small extracellular vesicles with a variable protein cargo in consonance with cell origin and pathophysiological conditions. Gestational diabetes mellitus (GDM) is characterized by different levels of chronic low-grade inflammation and vascular dysfunction; however, there are few data characterizing the serum exosomal protein cargo of GDM patients and associated signaling pathways. Eighteen pregnant women were enrolled in the study: 8 controls (CG) and 10 patients with GDM. Blood samples were collected from patients, for exosomes’ concentration. Protein abundance alterations were demonstrated by relative mass spectrometric analysis and their association with clinical parameters in GDM patients was performed using Pearson’s correlation analysis. The proteomics analysis revealed 78 significantly altered proteins when comparing GDM to CG, related to complement and coagulation cascades, platelet activation, prothrombotic factors and cholesterol metabolism. Down-regulation of Complement C3 (C3), Complement C5 (C5), C4-B (C4B), C4b-binding protein beta chain (C4BPB) and C4b-binding protein alpha chain (C4BPA), and up-regulation of C7, C9 and F12 were found in GDM. Our data indicated significant correlations between factors involved in the pathogenesis of GDM and clinical parameters that may improve the understanding of GDM pathophysiology. Data are available via ProteomeXchange with identifier PXD035673.

Due to their excellent mechanical and biocompatibility properties, titanium-based implants are successfully used as biomedical devices. However, when new bone formation fails for different reasons, impaired fracture healing becomes a clinical problem and affects the patient's quality of life. We aimed to design a new bioactive surface of titanium implants with a synergetic PEG biopolymer-based composition for gradual delivery of growth factors (FGF2, VEGF, and BMP4) during bone healing. The optimal architecture of non-cytotoxic polymeric coatings deposited by dip coating under controlled parameters was assessed both in cultured cells and in a rat tibial defect model (100% viability). Notably, the titanium adsorbed polymer matrix induced an improved healing process when compared with the individual action of each biomolecules. High-performance mass spectrometry analysis demonstrated that recovery after a traumatic event is governed by specific differentially regulated proteins, acting in a coordinated response to the external stimulus. Predicted protein interactions shown by STRING analysis were well organized in hub-based networks related with response to chemical, wound healing and response to stress pathways. The proposed functional polymer coatings of the titanium implants demonstrated the significant improvement of bone healing process after injury.