Size Exclusion Chromatography Fractions Research Articles

Abstract MP35: Angiotensin II-induced vascular response is enhanced by Extracellular Vesicles

Intro: The exact effects of extracellular vesicles (EVs) on renal hemodynamics and blood pressure remain to be determined. In this study, we examined the effect of EVs from hypertensive mice and humans on Angiotensin II (ANG II)-induced vascular response in an ex vivo model of isolated and perfused renal afferent arterioles (Af-Arts) and also assessed their effect on renal blood flow (RBF) and systolic blood pressure (SBP) in normotensive mice in vivo. Method: Circulating EVs were collected from mice with ANG II-induced hypertension (n=5) and hypertensive humans (n=2). Differential centrifugation (DCF) (5000G x 30min, followed by 17.000G for 30 minutes) was applied to generate an EV pellet (p20) which was subsequently run through size exclusion chromatography (SEC). EV size and concentration was determined by nanoparticle tracking analysis and spectral flow cytometry. Basic EV characterization was performed with cryo electron microscopy and western blotting of EV and non EV proteins. Following a 30-minute perfusion with EVs (p20 EV pellet (10 10 /ml) or fraction 1-3 or 4-6 of SEC (doses 1-9 7/ml )), dose-response curves to ANG II (10 -12 to 10 -6 mol/l) were measured in Af-Arts of C57BL/6 mice. Similar doses of EVs were infused into the penile vein of C57BL/6 mice and RBF and SBP were measured. As EVs might deliver AT1R we explored the final common pathway in vasoconstriction in vessels treated with EVs from hypertensive animals vs controls by performing immunoblotting of myosin light chain II S19 phosphorylation. Results: EVs deriving from ANG II-treated mice and hypertensive humans, but not those from normotensive control mice and humans, significantly enhanced the vasoconstrictor response to ANG II in Af-Arts of C57BL/6 mice. The same vasoconstrictor response was observed when Af-Arts were perfused with combined EV preps from DCF and SEC (fraction 1-3 vs fraction 1-6) from hypertensive animals. In vivo injection of EVs from ANG II-treated hypertensive mice reduced RBF and increased SBP in C57BL/6 mice compared to EVs from normotensive control mice. Immunoblotting of myosin light chain II S19 phosphorylation was inconclusive. Conclusion: These data suggest that EVs from hypertensive animals and humans directly affect ANG II response, renal hemodynamic and blood pressure regulation. Further work is needed to identify the EV effector candidate for enhanced ANG II response and possible effect on ART1 activation.

A Multiplexed SEC-MS Approach to Systematically Study the Interplay Between Protein Assembly-States and Phosphorylation Events.

Protein molecular interactions and post-translational modifications (PTMs), such as phosphorylation, can be co-dependent and reciprocally co-regulate each other. Although this interplay is central for many biological processes, a systematic method to simultaneously study assembly-states and PTMs from the same sample is critically missing. Here, we introduce SEC-MX (Size Exclusion Chromatography fractions MultipleXed), a global quantitative method combining Size Exclusion Chromatography and PTM-enrichment for simultaneous characterization of PTMs and assembly-states. SEC-MX enhances throughput, allows phosphopeptide enrichment, and facilitates quantitative differential comparisons between biological conditions. Applying SEC-MX to HEK293 and HCT116 cells, we generated a proof-of-concept dataset mapping thousands of phosphopeptides and their assembly-states. Our analysis revealed intricate relationships between phosphorylation events and assembly-states and generated testable hypotheses for follow-up studies. Overall, we establish SEC-MX as a valuable tool for exploring protein functions and regulation beyond abundance changes.

On-line coupling of hollow-fiber flow field-flow fractionation and depolarized multi-angle static light scattering (HF5/D-MALS). Proof of principle

Introduced here is the on-line coupling of hollow-fiber flow field-flow fractionation (HF5) to depolarized multi-angle static light scattering (D-MALS). HF5 is a size-based separation alternative to size-exclusion and hydrodynamic chromatography and asymmetric flow field-flow fractionation. HF5 can separate larger sizes than its chromatographic counterparts and provides several advantages over its fractionation counterpart, including reduced sample consumption and greater ease of operation. D-MALS is a variant of MALS in which the depolarized scattering from the analyte solution is measured at a variety of angles simultaneously. Measurements of depolarized scattering have previously been employed in studying the optical properties of solutions or suspensions, to determine the length of rod-like analytes, and to gain increased accuracy in the determination of analyte molar mass. The coupling HF5/D-MALS allows for the depolarization ratio of a solution or suspension to be measured continuously across the fractogram. This is demonstrated here for a Teflon latex the size range of which extends beyond that accessible to commercial size-exclusion columns. The results presented provide the first reported on-line HF5/D-MALS coupling, showing the feasibility of the technique as well as its realized potential for providing continuous depolarization measurements, inter alia.

SANS reveals lipid-dependent oligomerization of an intramembrane aspartyl protease from H. volcanii

Reactions that occur within the lipid membrane involve, at minimum, ternary complexes among the enzyme, substrate, and lipid. For many systems, the impact of the lipid in regulating activity or oligomerization state is poorly understood. Here, we used small-angle neutron scattering (SANS) to structurally characterize an intramembrane aspartyl protease (IAP), a class of membrane-bound enzymes that use membrane-embedded aspartate residues to hydrolyze transmembrane segments of biologically relevant substrates. We focused on an IAP ortholog from the halophilic archaeon Haloferax volcanii (HvoIAP). HvoIAP purified in n-dodecyl-β-D-maltoside (DDM) fractionates on size-exclusion chromatography (SEC) as two fractions. We show that, in DDM, the smaller SEC fraction is consistent with a compact HvoIAP monomer. Molecular dynamics flexible fitting conducted on an AlphaFold2-generated monomer produces a model in which loops are compact alongside the membrane-embedded helices. In contrast, SANS data collected on the second SEC fraction indicate an oligomer consistent with an elongated assembly of discrete HvoIAP monomers. Analysis of in-line SEC-SANS data of the HvoIAP oligomer, the first such experiment to be conducted on a membrane protein at Oak Ridge National Lab (ORNL), shows a diversity of elongated and spherical species, including one consistent with the tetrameric assembly reported for the Methanoculleus marisnigri JR1 IAP crystal structure not observed previously in solution. Reconstitution of monomeric HvoIAP into bicelles increases enzyme activity and results in the assembly of HvoIAP into a species with similar dimensions as the ensemble of oligomers isolated from DDM. Our study reveals lipid-mediated HvoIAP self-assembly and demonstrates the utility of in-line SEC-SANS in elucidating oligomerization states of small membrane proteins.

Analysis of aggregation profile of glucagon using SEC-HPLC and FFF-MALS methods.

Recently, the first generic glucagon for injection was approved for the treatment of severe hypoglycemia. Unlike its brand name recombinant glucagon, the generic glucagon is synthetic. Since glucagon has a high propensity to form aggregates in solution, it is essential to assess the aggregation profile of the synthetic glucagon compared to the recombinant glucagon. In this study, two robust separation methods, size-exclusion chromatography (SEC-HPLC) and field-flow fractionation coupled with a multi-angle light scattering detector (FFF-MALS), were employed to characterize generic and brand glucagon aggregation in six lots (three newly released, three expired). The presence of aggregation in samples was determined from the generated chromatograms and analyzed. The study showed that both products have comparable aggregation profiles. The SEC-HPLC demonstrated that in both glucagon versions, the expired lots had a higher percentage of dimers than the newly released lots, but even at expiration, the amount was negligible (∼0.1%). The FFF-MALS method did not detect any dimers or higher molecular weight aggregates. Further evaluation of the detection limit found that FFF-MALS was unable to detect aggregates at amounts lower than 0.5% of total glucagon. The negligible amounts of dimer detected in the generic and brand glucagon indicate that both versions are physically stable and are not prone to aggregation under clinically relevant conditions.

Global Interactome Mapping Reveals Pro-tumorigenic Interactions of NF-κB in Breast Cancer

NF-κB pathway is involved in inflammation; however, recent data shows its role also in cancer development and progression, including metastasis. To understand the role of NF-κB interactome dynamics in cancer, we study the complexity of breast cancer interactome in luminal A breast cancer model and its rearrangement associated with NF-κB modulation. Liquid chromatography-mass spectrometry measurement of 160 size-exclusion chromatography fractions identifies 5460 protein groups. Seven thousand five hundred sixty eight interactions among these proteins have been reconstructed by PrInCE algorithm, of which 2564 have been validated in independent datasets. NF-κB modulation leads to rearrangement of protein complexes involved in NF-κB signaling and immune response, cell cycle regulation, and DNA replication. Central NF-κB transcription regulator RELA co-elutes with interactors of NF-κB activator PRMT5, and these complexes are confirmed by AlphaPulldown prediction. A complementary immunoprecipitation experiment recapitulates RELA interactions with other NF-κB factors, associating NF-κB inhibition with lower binding of NF-κB activators to RELA. This study describes a network of pro-tumorigenic protein interactions and their rearrangement upon NF-κB inhibition with potential therapeutic implications in tumors with high NF-κB activity.

Unfolding of Lignin Structure Using Size-Exclusion Fractionation.

The heterogeneous and recalcitrant structure of lignin hinders its practical application. Here, we describe how new approaches to lignin characterization can reveal structural details that could ultimately lead to its more efficient utilization. A suite of methods, which enabled mass balance closure, the evaluation of structural features, and an accurate molecular weight (MW) determination, were employed and revealed unexpected structural features of the five alkali lignin fractions obtained with preparative size-exclusion chromatography (SEC). A thermal carbon analysis (TCA) provided quantitative temperature profiles based on sequential carbon evolution, including the final oxidation of char. The TCA results, supported with thermal desorption/pyrolysis gas chromatography-mass spectrometry (TD-Py-GC-MS) and 31P NMR spectroscopy, revealed the unfolding of the lignin structure as a result of the SEC fractionation, due to the disruption of the interactions between the high- and low-MW components. The "unraveled" lignin revealed poorly accessible hydroxyl groups and showed an altered thermal behavior. The fractionated lignin produced significantly less char upon pyrolysis, 2 vs. 47%. It also featured a higher occurrence of low-MW thermal evolution products, particularly guaiacol carbonyls, and more than double the number of OH groups accessible for phosphitylation. These observations indicate pronounced alterations in the lignin intermolecular association following size-exclusion fractionation, which may be used for more efficient lignin processing in biorefineries.

Analytical characterization of host-cell-protein-rich aggregates in monoclonal antibody solutions.

Host-cell proteins (HCPs) and high molecular weight (HMW) species have historically been treated as independent classes of impurities in the downstream processing of monoclonal antibodies (mAbs), but recent indications suggest that they may be partially linked. We have explored this connection with a shotgun proteomic analysis of HMW impurities that were isolated from harvest cell culture fluid (HCCF) and protein A eluate using size-exclusion chromatography (SEC). As part of the proteomic analysis, a cross-digest study was performed in which samples were analyzed using both the standard and native digest techniques to enable a fair comparison between bioprocess pools. This comparison reveals that the HCP profiles of HCCF and protein A eluate overlap substantially more than previous work has suggested, because hundreds of HCPs are conserved in aggregates that may be up to ~50 nm in hydrodynamic radius and that persist through the protein A capture step. Quantitative SWATH proteomics suggests that the majority of the protein A eluate's HCP mass is found in such aggregates, and this is corroborated by ELISA measurements on SEC fractions. The SWATH data also show that intra-aggregate concentrations of individual HCPs are positively correlated between aggregates that were isolated from HCCF and protein A eluate, and species that have generally been considered difficult to remove tend to be more concentrated than their counterparts. These observations support prior hypotheses regarding aggregate-mediated HCP persistence through protein A chromatography and highlight the importance of this persistence mechanism.

Defining the influence of size‐exclusion chromatography fraction window and ultrafiltration column choice on extracellular vesicle recovery in a skeletal muscle model

AbstractExtracellular vesicles (EVs) have the potential to provide new insights into skeletal muscle (SM) physiology and pathophysiology. However, current isolation protocols often do not eliminate co‐isolated components such as lipoproteins and RNA binding proteins that could confound outcomes and hinder downstream clinical translation. In this study, we validated an EV isolation protocol that combined size‐exclusion chromatography (SEC) with ultrafiltration (UF) to increase sample throughput, scalability and purity, while providing the very first analysis of the effects of UF column choice and fraction window on EV recovery. C2C12 myotube conditioned medium was pre‐concentrated using either Amicon® Ultra 15 or Vivaspin®20 100 KDa UF columns and processed by SEC (IZON, qEV 70 nm). The resulting thirty fractions obtained were individually analysed to identify an optimal fraction window for EV recovery. The EV marker TSG101 could be detected from fractions 5 to 14, while CD9 and Annexin A2 only up to fraction 6. ApoA1+ lipoprotein co‐isolates were detected from fraction 6 onwards for both protocols. Strikingly, Amicon and Vivaspin UF concentration protocols led to qualitative and quantitative variations in EV marker profiles and purity. Eliminating lipoprotein co‐isolation by reducing the SEC fraction window resulted in a net loss of particles, but increased measures of sample purity and had only a negligible impact on the presence of EV marker proteins. In conclusion, our study developed an effective UF+SEC protocol for the isolation of EVs based on sample purity (fractions 1–5) and total EV abundance (fractions 2–10). We provide evidence to demonstrate that the choice of UF column can affect the composition of the resulting EV preparation and needs to be considered when being applied in EV isolation studies in SM. The resulting protocols will be valuable in isolating highly pure EV preparations for applications in a range of therapeutic and diagnostic studies.

Cell-Free RNA from Plasma in Patients with Neuroblastoma: Exploring the Technical and Clinical Potential

Neuroblastoma affects mostly young children, bearing a high morbidity and mortality. Liquid biopsies, e.g., molecular analysis of circulating tumor-derived nucleic acids in blood, offer a minimally invasive diagnostic modality. Cell-free RNA (cfRNA) is released by all cells, especially cancer. It circulates in blood packed in extracellular vesicles (EV) or attached to proteins. We studied the feasibility of analyzing cfRNA and EV, isolated by size exclusion chromatography (SEC), from platelet-poor plasma from healthy controls (n = 40) and neuroblastoma patients with localized (n = 10) and metastatic disease (n = 30). The mRNA content was determined using several multiplex droplet digital PCR (ddPCR) assays for a neuroblastoma-specific gene panel (PHOX2B, TH, CHRNA3) and a cell cycle regulation panel (E2F1, CDC6, ATAD2, H2AFZ, MCM2, DHFR). We applied corrections for the presence of platelets. We demonstrated that neuroblastoma-specific markers were present in plasma from 14/30 patients with metastatic disease and not in healthy controls and patients with localized disease. Most cell cycle markers had a higher expression in patients. The mRNA markers were mostly present in the EV-enriched SEC fractions. In conclusion, cfRNA can be isolated from plasma and EV and analyzed using multiplex ddPCR. cfRNA is an interesting novel liquid biopsy-based target to explore further.

Laser scattering centrifugal liquid sedimentation method for the accurate quantitative analysis of mass-based size distributions of colloidal silica.

This paper proposes a laser scattering centrifugal liquid sedimentation (LS-CLS) method for the accurate quantitative analysis of the mass-based size distributions of colloidal silica. The optics comprised a laser diode light source and multi-pixel photon-counting detector for detecting scattered light intensity. The unique optics can only detect the light scattered by a sample through the interception of irradiated light. The developed centrifugal liquid sedimentation (CLS) method comprised a light-emitting diode and silicon photodiode detector for detecting transmittance light attenuation. The CLS apparatus could not accurately measure quantitative volume- or mass-based size distribution of poly-dispersed suspensions, such as colloidal silica, because the detecting signal includes both transmitted and scattered light. The LS-CLS method exhibited improved quantitative performance. Moreover, the LS-CLS system allowed the injection of samples with concentrations higher than that permitted by other particle size distribution measurement systems with particle size classification units using size-exclusion chromatography or centrifugal field-flow fractionation. The proposed LS-CLS method achieved an accurate quantitative analysis of the mass-based size distribution using both centrifugal classification and laser scattering optics. In particular, the system could measure the mass-based size distribution of approximately 20mgmL-1 poly-dispersed colloidal silica samples, such as in a mixture of the four mono-dispersed colloidal silica, with high resolution and precision, thereby demonstrating high quantitative performance. The measured size distributions were compared with those observed through transmission electron microscopy. The proposed system can be used in practical setups to achieve a reasonable degree of consistency for determining particle size distribution in industrial applications.

Investigating serum extracellular vesicles in Cystic Fibrosis

BackgroundExtracellular vesicles (EVs) are emerging as biomarkers of disease with diagnostic potential in CF. With the advent of highly effective modulator therapy, sputum production is less common and there is a need to identify novel markers of CF disease progression, exacerbation and response to therapies in accessible fluids such as serum. MethodsWe used size exclusion chromatography (SEC) to isolate and characterise EVs from the blood of PWCF of different ages and compared to ultracentrifugation (UC). We used nanoparticle tracking analysis to measure the number of EVs present in serum obtained from children and adults with CF. Mass spectrometry based proteomics was used to characterise protein expression changes between the groups. ResultsEVs were successfully isolated in SEC fractions from 250 µl serum from PWCF in greater numbers (p <0.01) than density ultracentrifugation. There was not a significant difference in EV numbers between young children with CF and controls. However, there was significantly more EVs in adults compared to children (<6yrs) (p < 0.05). EVs from PWCF before and after Kaftrio treatment were also analysed. Significant protein expression changes were observed within all 3 group. The largest changes detected were between children and adults with CF (57 proteins had a 1.5 fold change in expression with 19 significant changes p < 0.05) and PWCF taking Kaftrio (24 significant changes in EV protein expression was observed 12 months post treatment). ConclusionIn this pilot study, we performed an initial characterisation of EVs in serum from PWCF demonstrating the potential of serum EVs for further diagnostic investigation.

High-Throughput Proteome Profiling of Plasma and Native Plasma Complexes Using Native Chromatography.

We describe a high-throughput method for co-fractionation mass spectrometry (CF-MS) profiling for native plasma protein profiling. CF-MS allows the profiling of endogenous protein complexes between samples. Proteins often interact with other proteins and form macromolecular complexes that are different in disease states as well as cell states and cell types. This protocol describes an example for the sample preparation of 954 individual size exclusion chromatography (SEC) fractions, derived from 18 plasma samples that were separated into 53 fractions. Eighteen plasma samples were chosen based on the TMTpro multiplexing, but this methodology can be adapted for fewer or larger numbers of samples as appropriate. Our automated sample preparation method allows for high-throughput native plasma profiling, and we provide detailed methods for both a label-free and an isobaric labeling approach, discuss the merits of each approach, and detail the advantages of combining these strategies for comprehensive native plasma proteome profiling.

Thermal desorption gas chromatography-mass spectrometry for investigating the thermal degradation of polyurethanes.

Thermal Desorption Gas Chromatography-Mass Spectrometry (TD-GC-MS) was used to investigate the thermal degradation of two different polyurethanes (PUs). PU samples were heated at different heating rates and the desorbed products were collected in a cold injection system and thereafter submitted to GC-MS. The prospects and limitations of the detection and quantification of semi-volatile degradation products were investigated. Temperature dependent PU depolymerization was observed at temperatures above 200 °C, proved by an increased release of 1,4-butanediol and methylene diphenyl diisocyanate (MDI) representing the main building blocks of both polymers. Their release was monitored quantitatively based on external calibration with authentic compounds. Size Exclusion Chromatography (SEC) of the residues obtained after thermal desorption confirmed the initial competitive degradation mechanism indicating an equilibrium of crosslinking and depolymerization as previously suggested. Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry of SEC fractions of thermally degraded PUs provided additional hints on the degradation mechanism.

Large-scale top-down proteomics of the Arabidopsis thaliana leaf and chloroplast proteomes.

We present a large-scale top-down proteomics (TDP) study of plant leaf and chloroplast proteins, achieving the identification of over 4700 unique proteoforms. Using capillary zone electrophoresis coupled with tandem mass spectrometry analysis of offline size-exclusion chromatography fractions, we identify 3198 proteoforms for total leaf and 1836 proteoforms for chloroplast, with 1024 and 363 proteoforms having post-translational modifications, respectively. The electrophoretic mobility prediction of capillary zone electrophoresis allowed us to validate post-translational modifications that impact the charge state such as acetylation and phosphorylation. Identified modifications included Trp (di)oxidation events on six chloroplast proteins that may represent novel targets of singlet oxygen sensing. Furthermore, our TDP data provides direct experimental evidence of the N- and C-terminal residues of numerous mature proteoforms from chloroplast, mitochondria, endoplasmic reticulum, and other sub-cellular localizations. With this information, we suggest true transit peptide cleavage sites and correct sub-cellular localization signal predictions. This large-scale analysis illustrates the power of top-down proteoform identification of post-translational modifications and intact sequences that can benefit our understanding of both the structure and function of hundreds of plant proteins.

Determination of Short Chain Branching in LLDPE by Rheology

AbstractThe relationship between the structure and the final properties of materials is an important issue in polymer science. In particular, the properties of polyolefins are impacted by their branching contents. A simple rheological method is presented to estimate the degree of short chain branching (SCB) in linear low‐density polyethylene (LLDPE). A range of ethylene‐1‐hexene copolymers is synthetized using a metallocene catalyst to obtain homogenous LLDPE models with various comonomer contents. The polymers are analyzed by size exclusion chromatography, 13C NMR, and crystallization elution fractionation to determine both their molar masses and their chemical composition distributions. Subsequently, they are measured by rheology to assess the impact of SCB. This work provides calibration curves for rheology and proves that it is possible to predict the SCB content in LLDPE from the melt viscosity analysis.

Analysis of Host Cell Proteins in Monoclonal Antibody Therapeutics Through Size Exclusion Chromatography.

Host cell proteins (HCPs) are impurities derived from expression systems during the manufacturing of biotherapeutics. Even trace amounts of certain HCPs can potentially compromise product safety and quality. Therefore, comprehensive analytical characterization is necessary. In particular, understanding how each HCP co-purifies with the biotherapeutics throughout the purification process would help guide process development to avoid further contamination. We developed a new strategy based on size exclusion chromatography (SEC) fractionation followed by mass spectrometry (MS) analysis to study HCPs. Through an optimized experimental procedure, HCPs were effectively separated from monoclonal antibody (mAb) drug substances via SEC fractionation and sensitively detected with MS. Many HCPs were enriched in the high molecular weight fraction, thus indicating the formation of HCP-mAb complexes. SEC separation under mild denaturing conditions was demonstrated to disrupt weak interactions between certain HCPs and mAbs. The binding profiles of HCPs to mAbs were further characterized through comparison of the relative abundance of HCPs in each fraction under either native or mild denaturing SEC conditions. This new method not only achieves improved identification of HCPs in biotherapeutic drug substances but also offers an effective means to evaluate the binding properties between biotherapeutics and a wide range of HCPs.

Effect of cell culture media on extracellular vesicle secretion from mesenchymal stromal cells and neurons

BackgroundExtracellular vesicles (EVs) secreted by neuronal cells in vitro have promising therapeutic potential for brain diseases. Optimization of cell culture conditions and methodologies for high-yield isolation of EVs for preclinical and clinical applications, however, remains a challenge. ObjectiveTo probe the cell culture conditions required for optimal EV secretion by human-derived neuronal cells. MethodologyFirst, we optimized the EV purification protocol using human mesenchymal stromal cell (MSC) cultures. Next, we compared the effects of different variables in human pluripotent stem cell (hPSC)-derived neuronal cultures on EV secretion. EVs were isolated from cell conditioned media (CCM) and control media with no cells (NCC) using ultrafiltration combined with size-exclusion chromatography (SEC). The hPSC neurons were cultured in 2 different media from which EVs were collected at 2 maturation time-points (days 46 and 60). Stimulation with 25 mM KCl was also evaluated as an activator of EV secretion by neurons. The collected SEC fractions were analyzed by nanoparticle tracking analysis (NTA), protein concentration assay, and blinded transmission electron microscopy (TEM). ResultsA peak in cup-shaped particles was observed in SEC fractions 7–10 of MSC samples, but not corresponding media controls, indicating successful isolation of EVs. Culture medium had no significant effect on EV yield. The EV yield of the samples did not differ significantly according to the culture media used or the cell maturation time-points. Stimulation of neurons with KCl for 3 h reduced rather than increased the EV yield. ConclusionsWe demonstrated successful EV isolation from MSC and neuronal cells using an ultrafiltration-SEC method. The EV yield from MSC and neuronal cultures exhibited a large batch effect, apparently related to the culture media used, highlighting the importance of including NCC as a negative control in all cell culture experiments.

Investigation of the Multimer Cyclization Effect during Click Step-Growth Polymerization of AB-Type Macromonomers

In this work, by combining size exclusion chromatography (SEC) and liquid chromatography at the critical condition (LCCC) techniques, we have studied the multimer cyclization effect during click step-growth polymerization of AB-type macromonomers. First, by atom transfer radical polymerization (ATRP), we have synthesized a series of alkynyl and azide functional AB-type polystyrene macromonomers with different molar masses, i.e., PSn. Second, via the click step-growth polymerization of a PSn macromonomer in dimethylformamide (DMF) at varied reaction concentrations (Cs), we have prepared polyaddition products [(PSn)m] with large polydispersity indices, where m represents the average degree of polymerization of the macromonomer. Further, narrowly distributed fractions have been obtained by SEC fractionation. Third, we have quantitatively analyzed how the macromonomer molar mass, reaction concentration, and multimer molar mass influence the percentage of a cyclic topoisomer (wtcyclic) for (PSn)m samples. In particular, we have established the relation between wtcyclic and C for unfractionated (PSn)m, i.e., wtcyclic = 0.86–0.32 log C, and the relation between wtcyclic and m for fractionated (PSn)m prepared at a constant C = 100 g/L, i.e., log wtcyclic = −0.46m + 0.16. For the first time, our result has unambiguously pointed out that neither a cyclic topoisomer nor a linear topoisomer can be ignored when considering the structure–property quantitative relationship for polyaddition products.

Circulating extracellular vesicles provide valuable protein, but not DNA, biomarkers in metastatic breast cancer

AbstractDetection of cell‐free circulating tumour DNA (ctDNA) and cancer‐specific extracellular vesicles (EVs) in patient blood have been widely explored as non‐invasive biomarkers for cancer detection and disease follow up. However, most of the protocols used to isolate EVs co‐isolate other components and the actual value of EV‐associated markers remain unclear. To determine the optimal source of clinically‐relevant circulating biomarkers in breast cancer, we applied a size exclusion chromatography (SEC) procedure to analyse separately the content in nucleic acids of EV‐enriched and EV‐depleted fractions, in comparison to total plasma. Both cellular and mitochondrial DNA (cellDNA and mtDNA) were detected in EV‐rich and EV‐poor fractions. Analysing specific mutations identified from tumour tissues, we detected tumour‐specific cellular alleles in all SEC fractions. However, quantification of ctDNA from total plasma was more sensitive than from any SEC fractions. On the other hand, mtDNA was preferentially enriched in EV fractions from healthy donor, whereas cancer patients displayed more abundant mtDNA in total plasma, and equally distributed in all fractions. In contrast to nucleic acids, using a Multiplexed bead‐based EV‐analysis assay, we identified three surface proteins enriched in EVs from metastatic breast cancer plasma, suggesting that a small set of EV surface molecules could provide a disease signature. Our findings provide evidence that the detection of DNA within total circulating EVs does not add value as compared to the whole plasma, at least in the metastatic breast cancer patients used here. However, analysis of a subtype of EV‐associated proteins may reliably identify cancer patients. These non‐invasive biomarkers represent a promising tool for cancer diagnosis and real‐time monitoring of treatment efficacy and these results will impact the development of therapeutic approaches using EVs as targets or biomarkers of cancer.