Size Exclusion Chromatography Fractions Research Articles

Abstract B56: Neuroblastoma patient-derived tumor-specific mRNA and DNA in platelets and extracellular vesicles

Abstract Background: Neuroblastoma is the most common extracranial pediatric solid tumor. Current diagnostic strategies involve imaging and histopathology of tumor tissue and bone marrow. PHOX2B is a specific mRNA marker for neuroblastoma that can be used for detection of circulating tumor cells (CTCs) in peripheral blood and bone marrow. Methylated RASFF1A and ALK F1174L mutation can be used as a tumor-specific cell-free DNA marker. In this study, we aim to detect tumor-specific mRNA in neuroblastoma patient-derived platelets and plasma. Furthermore, we investigate if there is a diagnostic potential for extracellular vesicles (EV) as a source of neuroblastoma-specific mRNA and DNA. Method: Blood was collected in EDTA tubes and was centrifuged to separate platelets, platelet-free plasma (PFP), and mononuclear cells (MNC, consisting of leukocytes and CTCs). Using size exclusion chromatography (SEC), extracellular vesicles were isolated from 500uL PFP. The presence of EV was confirmed with Western blot, NanoSight, and electron microscopy. RNA was isolated from PFP, platelets, MNC, and SEC fractions using TRIzolTM Reagent. DNA was isolated using the QIAamp DNA Blood Mini Kit®. To detect neuroblastoma-specific RNA, PHOX2B and GUSB as reference gene was measured by RT-qPCR. To detect cell-free tumor DNA, methylated RASSF1A was detected with RT-qPCR, and the ALK F1174L mutation by digital droplet PCR. As a reference gene for DNA, albumin was used. Results: We collected blood from one healthy control and two patients with neuroblastoma (one at relapse and one in remission). GUSB could be detected in PFP, platelets, and MNC from the control and both patients. PHOX2B was detected in PFP, platelets, and MNC from the patient with a relapse and not in the control or other patient sample. EV were isolated from plasma from one healthy control and one patient at diagnosis. GUSB was present in vesicle-enriched fractions from both samples. PHOX2B was only detected in the neuroblastoma patient vesicle-enriched fractions. In vesicle-enriched fractions of the patient, more total DNA (albumin) was present than in the control. Tumor-specific DNA (ALK F1174L mutation and methylated RASSF1A) was predominantly present in the vesicle-poor, protein-rich fractions. Conclusion: This study demonstrates that neuroblastoma-specific mRNA and DNA can be detected in platelets, PFP and EV. In particular, mRNA is enriched in EV. This finding suggests that cell-free mRNA from plasma is conserved in EV and implies a role for cell-free RNA from liquid biopsies for diagnostic approaches. In the future, we will investigate the presence and prognostic importance of cell-free RNA and DNA in neuroblastoma and other solid tumors, especially those with few circulating tumor cells. Citation Format: Nathalie Lak, Lieke van Zogchel, Lily Zappeij-Kannegieter, Janine Stutterheim, Ellen van der Schoot, Lieve Tytgat. Neuroblastoma patient-derived tumor-specific mRNA and DNA in platelets and extracellular vesicles [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B56.

Identification of a compound, at a very low dose (less than 100 ng/g of diet), with lifespan-elongation activity towards SOD-1 mutant adults of Drosophila melanogaster in the hot water extract of Chlorella pyrenoidosa

Hot water extract of chlorella (WEC) increased the lifespan of superoxide dismutase (SOD)-1 mutant adults of Drosophila melanogaster in a dose dependent manner (200–800 µg/mL). Compounds in WEC were successively fractionated by solid phase extraction using a Sep-Pak C18 cartridge and size exclusion chromatography (SEC). Amino compounds in SEC fractions were derivatized with
 6-aminoquinolyl-N-hydroxylsuccinimidyl carbamate and analyzed by reversed phased-liquid chromatography-tandem mass spectrometry. Phenylalanine, phenethylamine, isopentylamine, and 2-methylbutylamine were identified in the SEC fraction, which increased the lifespan of the D. melanogaster mutant adults. Phenethylamine, at very low doses (6–60 ng/g of diet) that roughly corresponded to those of phenethylamine in WEC (200–800 μg/mL), increased the lifespan of the D. melanogaster adults, while isopentylamine did not exert the lifespan elongation activity. Since phenethylamine did not show SOD-like activity, it did not increase lifespan by direct antioxidant activity.

Fractionation and characterization of antioxidant peptides from rice bran protein hydrolysates stimulated by in vitro gastrointestinal digestion

AbstractBackground and objectiveRice bran protein concentrate was prepared and hydrolyzed by using pepsin–pancreatin. The obtained result was further filtered through 5‐kDa membranes. A permeate was collected and sequentially fractionated by different chromatographic techniques. The peptide fractions were determined for the antioxidant activities.FindingsThree peptide fractions were derived after separating the crude protein hydrolysates by anion‐exchange chromatography. Due to its highest ABTS radical scavenging and metal chelating activities (p < .05), fraction F2‐A was pooled and separated further by size‐exclusion chromatography (SEC). Among the SEC fractions, F1‐S showed strongest antioxidant activities; thus, it was collected for reverse‐phase HPLC purification. Subsequently, the peptide fractions were successfully separated and F4‐H had the greatest ABTS‐scavenging activities (214.61 µmol Trolox/g sample, respectively). In addition, it displayed the strongest ability to reduce ferric to ferrous ions (104.80 µmol FeSO4/g sample) (p < .05), while F1‐H showed the highest ability to chelate metal ions (266.40 µmol EDTA/g sample) (p < .05).ConclusionThese results demonstrate that hydrophobic amino acids play a major role in scavenging free radicals, whereas polar amino acids are responsible for reducing and chelating metal ions.Significance and noveltyRice bran protein composes of high ratio of hydrophobic amino acid. It is a suitable source of antioxidant peptides that might be produced during gastrointestinal digestion. Separation and purification using chromatographic techniques showed promising results for selectively fractionate antioxidant peptides with specific activity.

Isolation of Extracellular Vesicles from Leishmania spp.

Exosomes, a class of extracellular vesicles, are released by eukaryotes, bacteria, and archaea, as evident from both in vitro and in vivo studies. These nano-sized double-membraned vesicles play an important role in cell-to-cell communication, dysregulation of the immune system, and pathogenesis in a number of diseases, including leishmaniasis. Leishmania is a genus of obligate intracellular parasites, which infect host macrophages, are transmitted through the bite of a sandfly, and are shown to secrete exosomes with immunomodulatory activities. Given the importance of these vesicles in Leishmania spp. virulence, it is necessary to perform appropriate isolation and characterization in order to further study their relevance in the parasite's infectious life cycle. In this chapter, we describe four methods for the isolation of extracellular vesicles derived from Leishmania species including ultracentrifugation, polyethylene glycol-based precipitation, size-exclusion chromatography, and sucrose-gradient fractionation. Further, we describe the preparation of isolated samples for characterization by nanoparticle tracking analysis, transmission electron microscopy, and proteomic profiling.

Detergent-free isolation of native red blood cell membrane complexes

Over the past few decades, studies on the red blood cell (RBC) membrane gave rise to increasingly sophisticated although divergent models of its structural organization, since investigations were often performed in denaturing conditions using detergents. To access soluble isolated RBC membrane complexes with the preservation of their interactions and conformations, we decided to apply the recent SMALP (Styrene Maleic Acid Lipid Particles) technology to RBC ghosts. Depending on the ionic strength of buffers in which ghost membranes were resuspended, the isolated proteins within SMALPs could differ on Coomassie–stained gels, but with few changes when compared to ghost membrane SDS lysates. We subsequently produced SMALPs derived from ghosts from two different blood group phenotypes, RhD-positive and RhD-negative, both types of RBC expressing the RhCE proteins but only RhD-positive cells being able to express the RhD proteins. This allowed the isolation, by size exclusion chromatography (SEC), of soluble fractions containing the Rh complex, including the RhD protein or not, within SMALPs. The use a conformation-dependent anti-RhD antibody in immunoprecipitation studies performed on SEC fractions of SMALPs containing Rh proteins clearly demonstrated that the RhD protein, which was only present in SMALPs prepared from RhD-positive RBC ghosts, has preserved at least one important conformational RhD epitope. This approach opens new perspectives in the field of the erythroid membrane study, such as visualization of RBC membrane complexes in native conditions by cryo-electron microscopy (CryoEM) or immuno-tests with conformation-dependent antibodies against blood group antigens on separated and characterized SMALPs containing RBC membrane proteins.

Effect of peptidases secreted by the opportunistic pathogen Scedosporium aurantiacum on human epithelial cells.

Peptidases secreted by a clinical high-virulence Scedosporium aurantiacum isolate (strain WM 06.482; CBS 136046) under normoxic and hypoxic conditions were separated via size-exclusion chromatography, and peptidase activities present in each fraction were determined using class-specific substrates. The fractions demonstrating peptidase activity were assessed for their effects on the attachment and viability of A549 human lung epithelial cells in vitro. Of the peptidases detected in the size-exclusion chromatography fractions, the elastase-like peptidase reduced cell viability, the chymotrypsin-like peptidase was associated with cell detachment, and the cysteine peptidases were able to abolish both cell attachment and viability. The loss of cell viability and attachment became more prominent with an increase in the peptidase activity and could also be specifically prevented by addition of class-specific peptidase inhibitors. Our findings indicate that peptidases secreted by S. aurantiacum can breach the human alveolar epithelial cell barrier and, thus, may have a role in the pathobiology of the organism.

Stable native RIP9 complexes associate with C-to-U RNA editing activity, PPRs, RIPs, OZ1, ORRM1 and ISE2.

The mitochondrial and chloroplast mRNAs of the majority of land plants are modified through cytidine to uridine (C-to-U) RNA editing. Previously, forward and reverse genetic screens demonstrated a requirement for pentatricopeptide repeat (PPR) proteins for RNA editing. Moreover, chloroplast editing factors OZ1, RIP2, RIP9 and ORRM1 were identified in co-immunoprecipitation (co-IP) experiments, albeit the minimal complex sufficient for editing activity was never deduced. The current study focuses on isolated, intact complexes that are capable of editing distinct sites. Peak editing activity for four sites was discovered in size-exclusion chromatography (SEC) fractions ≥ 670kDa, while fractions estimated to be approximately 413kDa exhibited the greatest ability to convert a substrate containing the editing site rps14 C80. RNA content peaked in the ≥ 670kDa fraction. Treatment of active chloroplast extracts with RNase A abolished the relationship of editing activity with high-MW fractions, suggesting a structural RNA component in native complexes. By immunoblotting, RIP9, OTP86, OZ1 and ORRM1 were shown to be present in active gel filtration fractions, though OZ1 and ORRM1 were mainly found in low-MW inactive fractions. Active editing factor complexes were affinity-purified using anti-RIP9 antibodies, and orthologs to putative Arabidopsis thaliana RNA editing factor PPR proteins, RIP2, RIP9, RIP1, OZ1, ORRM1 and ISE2 were identified via mass spectrometry. Western blots from co-IP studies revealed the mutual association of OTP86 and OZ1 with native RIP9 complexes. Thus, RIP9 complexes were discovered to be highly associated with C-to-U RNA editing activity and other editing factors indicative of their critical role in vascular plant editosomes.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry with Activated Ion Electron Transfer Dissociation for Large-scale Top-down Proteomics.

Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as an efficient approach for top-down proteomics recently for its high-capacity separation and highly sensitive detection of proteoforms. However, the commonly used collision-based dissociation methods often cannot provide extensive fragmentation of proteoforms for thorough characterization. Activated ion electron transfer dissociation (AI-ETD), that combines infrared photoactivation concurrent with ETD, has shown better performance for proteoform fragmentation than higher energy-collisional dissociation (HCD) and standard ETD. Here, we present the first application of CZE-AI-ETD on an Orbitrap Fusion Lumos mass spectrometer for large-scale top-down proteomics of Escherichia coli (E. coli) cells. CZE-AI-ETD outperformed CZE-ETD regarding proteoform and protein identifications (IDs). CZE-AI-ETD reached comparable proteoform and protein IDs with CZE-HCD. CZE-AI-ETD tended to generate better expectation values (E values) of proteoforms than CZE-HCD and CZE-ETD, indicating a higher quality of MS/MS spectra from AI-ETD respecting the number of sequence-informative fragment ions generated. CZE-AI-ETD showed great reproducibility regarding the proteoform and protein IDs with relative standard deviations less than 4% and 2% (n = 3). Coupling size exclusion chromatography (SEC) to CZE-AI-ETD identified 3028 proteoforms and 387 proteins from E. coli cells with 1% spectrum level and 5% proteoform-level false discovery rates. The data represents the largest top-down proteomics dataset using the AI-ETD method so far. Single-shot CZE-AI-ETD of one SEC fraction identified 957 proteoforms and 253 proteins. N-terminal truncations, signal peptide cleavage, N-terminal methionine removal, and various post-translational modifications including protein N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation were detected.

Proteome Profiling of Exosomes Purified from a Small Amount of Human Serum: The Problem of Co-Purified Serum Components.

Untargeted proteomics analysis of extracellular vesicles (EVs) isolated from human serum or plasma remains a technical challenge due to the contamination of these vesicles with lipoproteins and other abundant serum components. Here we aimed to test a simple method of EV isolation from a small amount of human serum (<1 mL) using the size-exclusion chromatography (SEC) standalone for the discovery of vesicle-specific proteins by the untargeted LC–MS/MS shotgun approach. We selected the SEC fraction containing vesicles with the size of about 100 nm and enriched with exosome markers CD63 and CD81 (but not CD9 and TSG101) and analyzed it in a parallel to the subsequent SEC fraction enriched in the lipoprotein vesicles. In general, there were 267 proteins identified by LC–MS/MS in exosome-containing fraction (after exclusion of immunoglobulins), yet 94 of them might be considered as serum proteins. Hence, 173 exosome-related proteins were analyzed, including 92 proteins absent in lipoprotein-enriched fraction. In this set of exosome-related proteins, there were 45 species associated with the GO cellular compartment term “extracellular exosome”. Moreover, there were 31 proteins associated with different immune-related functions in this set, which putatively reflected the major role of exosomes released by immune cells present in the blood. We concluded that identified set of proteins included a bona fide exosomes components, yet the coverage of exosome proteome was low due to co-purified high abundant serum proteins. Nevertheless, the approach proposed in current work outperformed other comparable protocols regarding untargeted identification of exosome proteins and could be recommended for pilot exploratory studies when a small amount of a serum/plasma specimen is available.

Studying the bivariate tacticity distribution of poly-1-octene using two-dimensional liquid chromatography coupled with NMR

The application properties of poly-1-olefins depend on their molar mass and the microstructure i.e., tacticity. While average values for the latter are available from NMR, its distribution along and across the molar mass axis is not yet accessible. Two samples of poly-1-octene with different average tacticity were chosen for the investigation with liquid chromatography and off-line NMR detection. Because of the relatively low molar mass of the samples, liquid adsorption chromatography (LAC) is then a function of two parameters i.e., molar mass and tacticity. To study the tacticity distribution independent from the molar mass, samples were first preparatively fractionated by size-exclusion chromatography (SEC). Analysis of the SEC fractions showed a gradient of tacticity along the molar mass axis. The isolated SEC fractions were then preparatively separated by LAC using porous graphite as stationary phase. NMR analysis of the LAC fractions then revealed a fractionation according to tacticity. Notably, two distinct populations that differ in tacticity were shown, which may be a result of different polymerization pathways. A comparison of the experimental results with theoretical calculations confirms that the configuration of the macromolecules in solution determines the retention on the stationary phase in LAC.

Assembly of biopolymer particles after thermal conditioning of wheat bran proteins contained in a 21–43 kDa size exclusion chromatography fraction

Assembly of spherical nanoparticles after Ca2+-induced cold gelation of aqueous extracts of wheat bran was previously reported. Although nanospheres were suggested to be constituted by proteins and stabilized by calcium bridges, their exact origin was not fully demonstrated because of the complexity of wheat bran extracts. To deepen in this respect, by means of a bottom-up approach, the experiment was reproduced with fractions of wheat bran obtained by size exclusion chromatography. After thermal treatment and addition of CaCl2, fractions were characterized by UV–Vis spectroscopy, FTIR spectroscopy, SDS-PAGE electrophoresis, energy dispersive spectroscopy, differential scanning calorimetry, and electron microscopy. Instead of the expected nanospheres, spheroidal particles with diameters 210 – 330 nm were precipitated from a protein fraction of 21 – 43 kDa, even before the addition of CaCl2. Spectroscopic and SDS-PAGE characterization showed that particles were mainly constituted by proteins. Global evidence indicates that calcium did not participate in the assembly of the particles, but rather the driving force for protein aggregation was the denaturation caused by the heat treatment. According to their characteristics, spheroidal particles were classified as biopolymer particles.

A Top-Down Proteomics Platform Coupling Serial Size Exclusion Chromatography and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Mass spectrometry (MS) based top-down proteomics provides rich information about proteoforms arising from combinatorial amino acid sequence variations and post-translational modifications (PTMs). Fourier transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving power and provides high-accuracy mass measurements, presenting a powerful tool for top-down MS characterization of proteoforms. However, the detection and characterization of large proteins from complex mixtures remain challenging due to the exponential decrease in S: N with increasing molecular weight (MW) and coeluting low-MW proteins; thus, size-based fractionation of complex protein mixtures prior to MS analysis is necessary. Here, we directly combine MS-compatible serial size exclusion chromatography (sSEC) fractionation with 12 T FT-ICR MS for targeted top-down characterization of proteins from complex mixtures extracted from human and swine heart tissue. Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically resolved 31 distinct proteoforms (30-50 kDa) simultaneously in a single mass spectrum within a 100 m/ z window. Notably, within a 5 m/ z window, we obtained baseline isotopic resolution for 6 distinct large proteoforms (30-50 kDa). The ultrahigh resolving power of FT-ICR MS combined with sSEC fractionation enabled targeted top-down analysis of large proteoforms (>30 kDa) from the human heart proteome without extensive chromatographic separation or protein purification. Further separation of proteoforms inside the mass spectrometer (in-MS) allowed for isolation of individual proteoforms and targeted electron capture dissociation (ECD), yielding high sequence coverage. sSEC/FT-ICR ECD facilitated the identification and sequence characterization of important metabolic enzymes. This platform, which facilitates deep interrogation of proteoform primary structure, is highly tunable, allows for adjustment of MS and MS/MS parameters in real time, and can be utilized for a variety of complex protein mixtures.

Comparative analysis of the high molecular mass subproteomes of eight Bothrops snake venoms

Snake venoms are extremely active biological secretions composed primarily of various classes of enzymes. The genus Bothrops comprises various pit viper species that represent the most medically significant taxa in Central and South America, accounting for more human envenomations and fatalities than any other snakes in the region. Venom proteomes of many Bothrops species have been well-characterized but investigations have focused almost exclusively on proteins smaller than 100 kDa despite expression of larger components being documented in several Bothrops venoms. This study sought to achieve detailed identification of major components in the high molecular mass subproteome of venoms from eight Bothrops species (B. brazili, B. cotiara, B. insularis, B. jararaca, B. jararacussu, B. leucurus, B. moojeni and B. neuwiedi). Enzymes such as metalloproteinases and L-amino acid oxidases were the most prominent components identified in the first size-exclusion chromatography fractions of these venoms. Minor components also identified in the first peaks included 5′-nucleotidase, aminopeptidase, phosphodiesterase, and phospholipases A2 and B. Most of these components disappeared in electrophoretic profiles under reducing conditions, suggesting that they may be composed of more than one polypeptide chain. A significant shift in the molecular masses of these protein bands was observed following enzymatic N-deglycosylation, indicating that they may contain N-glycans. Furthermore, none of the identified high molecular mass proteins were shared by all eight species, revealing a high level of interspecific variability among these venom components.

Complex‐centric proteome profiling by SEC‐SWATH‐MS

Proteins are major effectors and regulators of biological processes that can elicit multiple functions depending on their interaction with other proteins. The organization of proteins into macromolecular complexes and their quantitative distribution across these complexes is, therefore, of great biological and clinical significance. In this paper, we describe an integrated experimental and computational technique to quantify hundreds of protein complexes in a single operation. The method consists of size exclusion chromatography (SEC) to fractionate native protein complexes, SWATH/DIA mass spectrometry to precisely quantify the proteins in each SEC fraction, and the computational framework CCprofiler to detect and quantify protein complexes by error‐controlled, complex‐centric analysis using prior information from generic protein interaction maps. Our analysis of the HEK293 cell line proteome delineates 462 complexes composed of 2,127 protein subunits. The technique identifies novel sub‐complexes and assembly intermediates of central regulatory complexes while assessing the quantitative subunit distribution across them. We make the toolset CCprofiler freely accessible and provide a web platform, SECexplorer, for custom exploration of the HEK293 proteome modularity.

Precipitation-based extracellular vesicle isolation from rat plasma co-precipitate vesicle-free microRNAs

ABSTRACTThe microRNA (miRNA) cargo contained in plasma extracellular vesicles (EVs) offers a relatively little explored source of biomarkers for brain diseases that can be obtained noninvasively. Methods to isolate EVs from plasma, however, are still being developed. For EV isolation, it is important to ensure the removal of vesicle-free miRNAs, which account for approximately two-thirds of plasma miRNAs. Membrane particle precipitation-based EV isolation is an appealing method because of the simple protocol and high yield. Here, we evaluated the performance of a precipitation-based method to obtain enriched EV-specific miRNAs from a small volume of rat plasma. We performed size-exclusion chromatography (SEC) on precipitation-isolated EV pellets and whole plasma. The SEC fractions were analysed using Nanoparticle Tracking Analysis (NTA), protein and miRNA concentration assays, and droplet digital polymerase chain reaction for four miRNAs (miR-142-3p, miR-124-3p, miR-23a, miR-122). Precipitation-isolated EVs and selected SEC fractions from the plasma were also analysed with transmission electron microscopy (TEM). Precipitation-based EV isolation co-precipitated 9% to 15% of plasma proteins and 21% to 99% of vesicle-free miRNAs, depending on the individual miRNAs. In addition, the amount of miR-142-3p, found mainly in EV fractions, was decreased in the EV fractions, indicating that part of it was lost during precipitation-based isolation. Western blot and TEM revealed both protein and lipoprotein contamination in the precipitation-isolated EV-pellets. Our findings indicate that a precipitation-based method is not sufficient for purifying plasma EV-contained miRNA cargo. The particle number measured by NTA is high, but this is mostly due to the contaminating lipoproteins. Although a part of the vesicle-free miRNA is removed, vesicle-free miRNA still dominates in plasma EV pellets isolated by the precipitation-based method.

Size exclusion chromatography – Inductively coupled plasma – Mass spectrometry for determining metal-low molecular weight compound complexes in natural wines

Size exclusion chromatography (SEC) hyphenated to inductively coupled plasma – mass spectrometry (ICP-MS), as a specific detector for metals, has been used for monitoring and determining metal-low molecular weight organic compound (LMWC) complexes in natural wines. SEC with UV detection (wavelength of 205 nm) was used for monitoring organic compounds eluted from the chromatographic column. SEC-UV has revealed the presence of low molecular weight compounds (commonly three fractions of molecular weights ranging from 230 to 1579 Da).’ Further experiments using ICP-MS as a detector showed that elements such as B, Cu, Li, Mn, Ni, Ti, and Zn are bound to compounds of molecular weights within the 338–1579 Da range. Total metal concentrations, as well as metal concentrations in SEC fractions were also assessed in several monovarietal red (five varieties) and monovarietal white (three varieties) wines.

HSP70 Copurifies with Zika Virus Particles

Zika virus (ZIKV) has been identified as a cause of neurologic diseases in infants and Guillain-Barré Syndrome, and currently, no therapeutics or vaccines are approved. In this study, we sought to identify potential host proteins interacting with ZIKV particles to gain better insights into viral infectivity. Viral particles were purified through density-gradient centrifugation and subsequently, size-exclusion chromatography (SEC). Mass spectrometric analyses revealed viral envelope protein and HSP70 to comigrate in only one SEC fraction. Neither of these proteins were found in any other SEC fractions. We then performed neutralization assays and found that incubating viral particles with antibody against HSP70 indeed significantly reduced viral infectivity, while HSC70 antibody did not. Preincubating cells with recombinant HSP70 also decreased viral infectivity. Knockdown and inhibition of HSP70 also significantly diminished viral production. These results implicate HSP70 in the pathogenesis of ZIKV and identify HSP70 as a potential host therapeutic target against ZIKV infection.

Analysis of Human Nuclear Protein Complexes by Quantitative Mass Spectrometry Profiling.

Analysis of protein complexes provides insights into how the ensemble of expressed proteome is organized into functional units. While there have been advances in techniques for proteome-wide profiling of cytoplasmic protein complexes, information about human nuclear protein complexes are very limited. To close this gap, we combined native size exclusion chromatography (SEC) with label-free quantitative MS profiling to characterize hundreds of nuclear protein complexes isolated from human glioblastoma multiforme T98G cells. We identified 1794 proteins that overlapped between two biological replicates of which 1244 proteins were characterized as existing within stably associated putative complexes. co-IP experiments confirmed the interaction of PARP1 with Ku70/Ku80 proteins and HDAC1 (histone deacetylase complex 1) and CHD4. HDAC1/2 also co-migrated with various SIN3A and nucleosome remodeling and deacetylase components in SEC fractionation including SIN3A, SAP30, RBBP4, RBBP7, and NCOR1. Co-elution of HDAC1/2/3 with both the KDM1A and RCOR1 further confirmed that these proteins are integral components of human deacetylase complexes. Our approach also demonstrated the ability to identify potential moonlighting complexes and novel complexes containing uncharacterized proteins. Overall, the results demonstrated the utility of SEC fractionation and LC-MS analysis for system-wide profiling of proteins to predict the existence of distinct forms of nuclear protein complexes.

616: Comparison of circulating extracellular vesicle versus plasma proteins for prediction of spontaneous preterm birth

Extracellular vesicles (EVs) provide a novel and increasingly useful source of proteomic information separate from protein analysis in plasma alone. We hypothesize that EV protein profiling and analysis will provide a unique group of protein biomarkers for spontaneous preterm births compared to direct analysis of free plasma proteins. Twenty-five sPTB and 50 term EDTA plasma specimens were obtained at a median of 25 weeks gestation and matched by maternal age, race and gestational age at sampling. Vesicles were isolated from one aliquot via size exclusion chromatography (SEC) and the other was left as plasma (PPS). Both samples were analyzed by multiple reaction monitoring mass spectrometry for a targeted set of proteins. Data from the SEC and PPS samples was analyzed with a bootstrap receiver-operator curve (ROC) analysis that was performed with label permutation for a false discovery rate <20% for the identification of sPTB. The permutation test of Venkatraman was used to compare the significance of the area under the curve (AUC) in the SEC and PPS fractions for each of the top paired candidate markers. Of the 132 proteins evaluated, the median relative concentrations in the plasma versus EV samples for each protein are presented in the figure which illustrate little similarity between the fractions. The top ten bootstrapped AUCs, with regard to the prediction of sPTB, are presented in Table 1. The AUCs among the SEC were significantly higher than the corresponding PPS with no overlap in the top performing proteins between the two fractions. Among the top single marker predictors of sPTB from both EV and plasma proteins, the EV protein markers tended to display significantly better ROC characteristics. Circulating EV associated proteins represent a source of biomarker information that is distinct from proteins derived directly from the plasma.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Confounding factors in vesicle uptake studies using fluorescent lipophilic membrane dyes

ABSTRACTSmall extracellular vesicles (sEVs) such as exosomes are nanocarriers of proteins, RNAs and DNAs. Isolation of pure sEV populations remains challenging, with reports of protein and lipoprotein contaminants in the isolates. Cellular uptake – a cornerstone for understanding exosome and sEV function – is frequently examined using lipophilic dyes such as PKH67 or CellMask to label the vesicles. In this study, we investigated whether contaminants can confound the outcomes from sEV and exosomes uptake experiments. sEVs were isolated from blood plasma of fasted or non-fasted rats as well as from serum-supplemented or serum-free conditioned cell culture medium using size-exclusion chromatography (SEC). Eluent fractions were characterized using nanoparticle tracking, protein and triglyceride assays and immunoassays. SEC fractions were labelled with different lipophilic dyes and cellular uptake was quantified using endothelial cells or primary cardiomyocytes. We report co-isolation of sEVs with apolipoprotein B-containing lipoproteins. Cellular dye transfer did not correspond to sEV content of the SEC fractions, but was severely affected by lipoprotein and protein content. Overnight fasting of rats decreased lipoprotein content and also decreased dye transfer, while late, sEV-poor/protein-rich fractions demonstrated even greater dye transfer. The potential for dye transfer to occur in the complete absence of sEVs was clearly shown by experiments using staining of sEV-depleted serum or pure protein sample. In conclusion, proteins and lipoproteins can make a substantial contribution to transfer of lipophilic dyes to recipient cells. Considering the likelihood of contamination of sEV and exosome isolates, lipophilic dye staining experiments should be carefully controlled, and conclusions interpreted with caution.