Borges, Chad R.

Extracellular vesicles (EVs) play important roles in (patho)physiological processes by mediating cell communication. Although EVs contain glycans and glycosaminoglycans (GAGs), these biomolecules have been overlooked due to technical challenges in comprehensive glycome analysis coupled with EV isolation. Conventional mass spectrometry (MS)-based methods are restricted to the assessment of N-linked glycans. Therefore, methods to comprehensively analyze all glyco-polymer classes on EVs are urgently needed. In this study, tangential flow filtration-based EV isolation was coupled with glycan node analysis (GNA) as an innovative and robust approach to characterize most major glyco-polymer features of EVs. GNA is a molecularly bottom-up gas chromatography-MS technique that provides unique information that is unobtainable with conventional methods. The results indicate that GNA can identify EV-associated glyco-polymers that would remain undetected with conventional MS methods. Specifically, predictions based on GNA identified a GAG (hyaluronan) with varying abundance on EVs from two different melanoma cell lines. Enzyme-linked immunosorbent assays and enzymatic stripping protocols confirmed the differential abundance of EV-associated hyaluronan. These results lay the framework to explore GNA as a tool to assess major glycan classes on EVs, unveiling the EV glycocode and its biological functions.

Blood plasma is a readily accessible source of extracellular vesicles (EVs), i.e., cell-secreted nanosized carriers that contain various biomolecules, including glycans. Previous studies have demonstrated that glycans play a major role in physiological and pathological processes, and certain plasma glycans have been associated with disease conditions. However, glycome studies have been limited by a lack of analytical techniques with the throughput capacity necessary to study hundreds of clinical samples. This study is the first to characterize the EV plasma glycome based on all major glycan classes. The results based on glycan node analysis revealed, as expected, that plasma-derived EVs have distinct glycan features from donor-matched whole plasma. Specifically, glycan nodes corresponding to those observed in chondroitin sulfate, dermatan sulfate, type I keratan sulfate, and type II keratan sulfate were enriched on EVs. The identification of specific differences in glycan features in plasma vs. plasma-derived EVs is relevant for understanding the physiological role of EVs and as a reference for future diagnostic studies. Additionally, the results indicate that EV glycan nodes do not substantially differ among a small set of healthy donors. These results lay the framework for the further evaluation of all EV glycan classes as diagnostic markers, therapeutic targets, and biologically active components in health and disease.

AbstractBackground: Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1–84) and N-terminally truncated PTH (PTH7–84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminal PTH variants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1–84] and 2 N-terminal variants, aa7–84 and aa34–84.Methods: Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards.Results: Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28–84, aa48–84, aa34–77, aa37–77, and aa38–77. Quantitative SRM assays were developed for PTH1–84, PTH7–84, and variant aa34–84. Peptides exhibited linear responses (R2 = 0.90–0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16–31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3%–12% for all peptides, with <5% chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5%–9%.Conclusions: Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH.