Size Exclusion Chromatography Separation Research Articles

Quantitative description of the quality of size exclusion chromatography separations

Size exclusion chromatography (SEC) is unique among chromatographic methods as it allows separation of non-retained analytes. However, such mechanism often put an analytical scientist in front of relatively poorly resolved set of peaks that may have strikingly different abundance. The description of such chromatograms needs a particular approach to accurately capture the overall quality of separation. Consequently, use of a single parameter description may not be accurate enough and therefore we introduce a dimensionless separation quality factor, which is based on five SEC specific measures (peak-to-valley, elution window width, peak widths, peak-positioning and recovery). Combining several factors allowed detailed differentiation of various simulated separations, clearly correlating column characteristics with specific contributions to separation quality whether they concern a single peak pair or entire peak landscape. The method could be further elaborated by the addition of normalized priority weighting allowing for flexible quality quantification of a relevant portion of real-life nucleic acid separation on different columns. With growing complexity of biotherapeutics to be separated, such a term is predicted to be a useful response function for purposes of factorial method optimization.

Protein factors of capacitation and decapacitation in canine seminal plasma

The aim of the present study was to determine the proteins in canine seminal plasma with direct effect on specific sperm parameters during fertilisation. The influence of seminal plasma proteins was focused on three processes: capacitation, hyperactivation and decapacitation. Size exclusion chromatographic fractional separation of proteins from seminal plasma based on their molecular weights was performed. In addition, two-dimensional electrophoresis of two seminal protein fractions was done. The effect of protein molecules on the motion and kinetic parameters of the male gametes in in vitro conditions was monitored by computer-assisted sperm analysis. The activity of alkaline phosphatase, creatine kinase and alanine aminotransferase in whole seminal plasma and each chromatography fraction was measured spectrophotometrically using the BA-88A semi-automatic chemistry analyzer. The protein fractions with different molecular weights affected the in vitro motility and kinetic characteristics (curvilinear velocity, straightness, linearity and amplitude of lateral head displacement) of spermatozoa, leading to hyperactivation or initiating decapacitation kinetics changes. The detailed study of the seminal plasma proteome would add fundamental information about the processes associated with physiological changes occurring in spermatozoa before fertilisation. The study of the canine seminal plasma proteome could add relevant information about its effects on the fertilising ability of the male gametes and the changes occurring in them before fertilisation.

Monomethylarsonous acid binds to Cys-104α and Cys-112β of hemoglobin in acute promyelocytic leukemia patients treated with arsenic trioxide

Arsenic trioxide (As2O3) has prominent effect in treating acute promyelocytic leukemia (APL). Identification of arsenic-binding proteins has gained attention for their important biological functions. However, none has been published concerning the binding mechanism of arsenic with hemoglobin (Hb) in APL patients after treatment of As2O3. The present study discloses the binding sites of arsenic on Hb in APL patients. Concentrations of inorganic arsenic (iAs), monomethyl arsenic (MMA), and dimethyl arsenic (DMA) in erythrocytes of APL patients were quantified using HPLC-inductively coupled plasma-mass spectroscopy (HPLC-ICP-MS). Hb-bound arsenic was identified by size-exclusion chromatography ICP-MS. The binding sites of arsenic on Hb were determined by mass spectrometry (MS). The concentration trend of arsenic species in erythrocytes of 9 APL patients treated with As2O3 was iAs>MMA>DMA, and MMA was the predominant methylated arsenic metabolite. Size-exclusion chromatography separation of free and protein-bound arsenic by simultaneous monitoring of 57Fe and 75As demonstrated the presence of Hb-bound arsenic. MS information suggested monomethylarsonous (MMAIII) was the dominant arsenic bound to Hb, and further identified that Cys-104α and Cys-112β were two binding sites of MMAIII in Hb. MMAIII binding to Cys-104α and Cys-112β was responsible for arsenic accumulation in erythrocytes of APL patients. This interaction may contribute to understand the therapeutic effect of As2O3 as an anticancer drug and its toxicity on APL patients.

Biochemical separation of Cetuximab-Fab from papain-digested antibody fragments and radiolabeling with 64Cu for potential use in radioimmunotheranostics

Engineered Fab fragments of monoclonal antibodies (mAbs) after radiolabeling with suitable radiometals have the potential to play a key role in personalized radioimmunotheranostics of cancer patients. In this study, we have generated Fab fragment of Cetuximab, a mAb targeting epidermal growth factor receptor (EGFR) expression and purified from the Fc and other fragments by ultrafiltration and affinity chromatography. The Cetuximab-Fab was conjugated with a suitable bifunctional chelator and radiolabeled with no-carrier-added (NCA) 64Cu produced via 64Zn (n, p) 64Cu reaction in a nuclear reactor. The radioimmunoconjugate obtained after size exclusion chromatographic separation possessed >95% radiochemical purity and it retained its integrity over at least three half-lives of the radiometal. Biodistribution studies was performed in fibrosarcoma tumor bearing Swiss mice, which demonstrated the explicit need for purification of the Cetuximab-Fab from Fc fragments. Enhanced and rapid tumor uptake with decent tumor-to-background ratio with prolonged retention was observed when radiolabeled purified Cetuximab-Fab was intravenously administered in animal models. Overall, this preclinical study established the pivotal role of separation science and technology to obtain the radioimmunoconjugate with requisite purity in order to demonstrate optimal pharmacokinetics and maximized treatment efficacy.

Multi-Functional Separation Mode-Ion Chromatography Using L-Pyroglutamic Acid Eluent for Simultaneous Determination of Sugars, Organic Acids, and Ethanol during Multiple Parallel Fermentation of Rice Wine

An effective evaluation method for understanding fermentation is required to support the implementation of newly developed yeast strains in brewing. Our group developed a multi-functional separation mode-ion chromatography (MFS-IC) with conductivity and refractive index detectors to separate key species (glucose, ethanol, and organic acids: pyruvate, citrate, L-malate, and succinate) with ethyl α-D-glucoside and glycerol to understand the fermentation behavior in brewing. Optimal chromatographic resolutions for determining eight different key species in rice wine by the MFS-IC were obtained in 45 min on low-molecular size-exclusion chromatographic separation columns modified with a strong cation-exchanger as the stationary phase with an eluent of L-pyroglutamic acid. Understanding the behavior of analytes in rice wine samples during rice wine fermentation enables the monitoring of the progress of saccharification, glycolysis, ethanol fermentation, and the tricarboxylic acid (TCA) cycle. These results are expected to contribute to understanding fermentation characteristics and implementing newly developed yeast strains in the fermentation field.

NOM fractionation by HPSEC-DAD-OCD for predicting trihalomethane disinfection by-product formation potential in full-scale drinking water treatment plants

Chlorination is a common method for water disinfection; however, it leads to the formation of disinfection by-products (DBPs), which are undesirable toxic pollutants. To prevent their formation, it is crucial to understand the reactivity of natural organic matter (NOM), which is considered a dominant precursor of DBPs. We propose a novel size exclusion chromatography (SEC) approach to evaluate NOM reactivity and the formation potential of total trihalomethanes-formation potentials (tTHMs-FP) and four regulated species (i.e. CHCl3, CHBrCl2, CHBr2Cl, and CHBr3). This method combines enhanced SEC separation with two analytical columns working in tandem and quantification of apparent molecular weight (AMW) NOM fractions using C content (organic carbon detector, OCD), 254-nm spectroscopic (diode-array detector, DAD) measurements, and spectral slopes at low (S206–240) and high (S350–380) wavelengths. Links between THMs-FP and NOM fractions from high performance size exclusion chromatography HPSEC-DAD-OCD were investigated using statistical modelling with multiple linear regressions for samples taken alongside conventional full-scale as well as full- and pilot-scale electrodialysis reversal and bench-scale ion exchange resins. The proposed models revealed promising correlations between the AMW NOM fractions and the THMs-FP. Methodological changes increased fractionated signal correlations relative to bulk regressions, especially in the proposed HPSEC-DAD-OCD method. Furthermore, spectroscopic models based on fractionated signals are presented, providing a promising approach to predict THMs-FP simultaneously considering the effect of the dominant THMs precursors, NOM and Br−.

Development of native mass spectrometry with nanoelectrospray ionization coupled to size exclusion chromatography for proteins.

Native mass spectrometry (MS) is an analytical technique used to determine the molecular mass of protein complexes without cross-linking. Size exclusion chromatography (SEC) coupled with native MS using conventional electrospray ionization (ESI) has been reported to allow online buffer exchange. To detect a wide variety of protein complexes without a collapse in the ionization process, it is important to build an online system that enables robust analysis with a low flow rate. We created an online native MS system equipped with nanoESI connected to the SEC component (online SEC/nanoESI system) and optimized several parameters for SEC separation and ionization. The constructed system was used to measure a solution consisting of a protein mixture of various molecular masses (10-300 kDa) to verify characteristics such as the measurable molecular mass range, reproducibility, and online buffer exchange. The optimal flow rates for SEC separation and nanoESI analysis using this system were 200 and 1μL/min, respectively. This system was able to analyze proteins in the ranges of 10-300 and 20-300 kDa for protein samples in ammonium acetate and nonvolatile buffer, respectively. Furthermore, the results of consecutive measurements showed that the relative standard deviations of the retention times and observed masses for each protein were sufficiently small. We created an online SEC/nanoESI system and evaluated its utility for the analysis of various proteins in conventional measurement solvent and nonvolatile buffer. As a result, the structural stability and resolution of the proteins were found to be sufficient when using online buffer exchange. Therefore, this online SEC/nanoESI system would be a useful technique for obtaining mass spectra of various proteins automatically with good resolution, simply by loading samples into an autosampler.

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.

Size exclusion chromatography using large pore size media induces adverse conformational changes of inactivated foot-and-mouth disease virus particles

Size exclusion chromatography (SEC) of biomacromolecules using large pore size media and long column are usually necessary to obtain a satisfactory separation. However, the SEC separation of inactivated foot and mouth disease virus (FMDV) was found to induce some subtle but important conformational changes of FMDV in a pore-size and column length dependent manner. Here three Sephacryl media including S-300 HR, S-400 HR, and S-500 HR were tested, whose pore sizes were smaller than, similar to, and larger than the FMDV particles, respectively. High performance size exclusion chromatography (HPSEC) analyses showed that the FMDV after all these three SEC processes had earlier retention time, compared with that before SEC, but had no detectable difference in particle integrity. Longer SEC column led to more significant peak shifting in subsequent HPSEC analysis of FMDV. Further analyses indicated the SEC using larger pore size media induced more remarkable conformational changes and decrease in thermostability of FMDV, as well as decrease in immunogenicity in animal test. Fluorescence probe diffusion study suggested compared to SEC by S300, the compactness of the viral capsid after SEC by S400 and S500 was decreased, possibly due to more shear-induced FMDV particle rotation and inter-particle collision inside the media pores, as well as their interactions with the pore walls of the media during flowing through the column. Finally, a stabilization strategy by appending 5 mM CaCl2 in mobile phase of SEC separation was proposed and proved to efficiently maintain the conformation of the FMDV.

Abstract 10979: Liver-Humanized Mice Provide a New Platform to Study Human Cardiometabolic Diseases

Introduction: Conventional mouse models do not fully recapitulate human cardiometabolic diseases (CMD), e.g. , atherosclerosis and nonalcoholic steatohepatitis (NASH), due to species differences in metabolism. Liver-humanized mice (LHM), i.e. , mice repopulated with human hepatocytes, have been shown to be an improved translatable model to study human hepatic and lipoprotein metabolism. Hypothesis: As dietary challenge is often used to induce CMD in animal models, we sought to characterize lipoprotein and lipid metabolism in LHM challenged by different diets. Methods: Immune-deficient FRGN mice receiving hepatocytes from different human donors (LHM) or NOD mouse (liver-murinized mice, LMM) were fed a high-fat/high-sucrose diet (HFHSD) for 8 weeks or a high-fat/high-fructose/high-cholesterol diet (NASH-diet) for 8, 12, 16 or 20 weeks. Plasma lipids and lipoproteins were quantified after size-exclusion chromatography separation or by routine clinical assays. Cholesterol in the descending aorta was analyzed by GC-MS. Histologic analysis of hearts or livers was done using hematoxylin/eosin or Oil Red O staining, and anti-mouse CD68-antibody was used to detect murine macrophages. Results: Compared with LMM, LHM fed either HFHSD or NASH-diet accumulated plasma cholesterol and triglycerides within LDL and triglyceride-rich particles (VLDL and remnants). However, only the NASH-diet induced a notable hyperlipidemia and a substantial increase in cholesteryl esters within the aorta. NASH-diet also determined a clear accumulation of neutral lipids and CD68-positive cells in the aortic valves of LHM. Similarly, hepatocyte ballooning, liver steatosis and fibrosis were also induced by the NASH-diet. All these pathologic changes occurred during diet challenge at different time points. Conclusions: LHM developed severe hyperlipidemia, atherosclerosis and NASH when fed a NASH-diet, but not on HFHSD. The high quantity of cholesterol in the NASH-diet likely compensates for the high utilization of cholesterol of LHM due to their known increase in bile acid synthesis, prompting dyslipidemia and CMD. LHM fed a NASH-diet is an improved model for human CMD and for the development of therapeutic strategies rapidly translatable to the human condition.

Fast determination of functionality-type × molecular-weight distribution of propoxylates with varying numbers of hydroxyl end-groups using gradient–normal-phase liquid chromatography × ultra-high pressure size-exclusion chromatography

Understanding the relation between chemical characteristics and properties of synthetic polymers is one of the challenges faced by analytical chemists in industry. This is a complex task, as polymers are not synthesized as single molecule, but are populations of chemically similar compounds with distributions over several properties. The latter include, for example, molecular weight, nature of end-groups (functionality), and chemical composition.In this paper, comprehensive two-dimensional liquid chromatography was used to determine the combined functionality-type and molecular-weight distributions of hydroxy‑functionalized propoxylates. Propoxylates derived from different initiators (one up to eight terminal hydroxyl groups) were separated in the first dimension using a gradient normal-phase LC separation (NPLC). In the second dimension ultra-high pressure size-exclusion chromatography separation (UHPSEC), further speciating distributions based on molecular size. The developed NPLC × SEC method with evaporative light-scattering detection can be used for the fast screening (< 30 min) of mutually dependent functionality-type and molecular-weight distributions of unknown propoxylates.

Characterization of ultrahigh molecular weight poly(ethylene oxide) by size-exclusion chromatography with multiangle light scattering detection

This study investigated the experimental conditions needed to obtain molecular weight distribution (MWD) of ultrahigh MW poly(ethylene oxide) (PEO) using size exclusion chromatography (SEC) hyphenated with a multiangle light scattering photometer (MALS) and a differential refractive index detector (RI). Ultrahigh MW components yielded non-linear angular dependency in scattered light intensities. The first-order linear fitting using Zimm formalism resulted in significant differences in MW depending on if the signals from detector 4 to 16 were used in the fitting or only five low-angles from 4 to 8 were used. In contrast, no significant differences in MW were obtained for lower MW PEO samples (equal or less than 1000 KDa) between the two fitting approaches. It was thus proposed to use only the five low-angles to derive MW for a sample with broad polydispersity including both ultrahigh and low MW components. The SEC separation was done using one column designed for ultrahigh MW polymer separation connected with another mixed-bed column. The ultrahigh MW column allowed separation and characterization of polymeric components in the MW range between 10 and 50 million Dalton (MDa) and the size range between 300 and 600 nm in radius of gyration (Rg). Online calibration curves were obtained from the linear fittings of MW as a function of elution volume. MW polydispersity was derived from the online calibration curve showing that the ultrahigh MW PEO had higher polydispersity than the lower MW samples. The double logarithmic plot of radius of gyration versus MW indicated that both ultrahigh MW and low MW PEO would adopt expanded coil conformations in the aqueous solution.

Pepsin Hydrolysis of Orange By-Products for the Production of Bioactive Peptides with Gastrointestinal Resistant Properties

Recently, the use of bioactive compounds in improving human health has received more attention. The aim of the present study was to hydrolyze orange seed proteins using pepsin enzyme to obtain bioactive peptides as well as to study the stability of such activity after simulated gastrointestinal digestion conditions. The method was optimized using different enzyme concentrations from 1% to 3%, hydrolysis times between 2 and 5 h, and an optimal temperature of 33 °C. Biological activities including α-glucosidase inhibition, α-amylase inhibition, Angiotensin I-Converting Enzyme (ACEI) inhibition, ferric reducing antioxidant power, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity were evaluated. According to the results, a significant higher value of the biological activity (p < 0.05) was observed using an enzyme ratio of 0.03 E/S and hydrolysis time of 3.5 h. After size-exclusion chromatography separation, fractions 45–49 and 50–54 showed the highest biological roles such as antioxidant, ACEI inhibitory, and hypoglycemic. Fractions with the highest biological activity were purified using RP-HPLC and analyzed using nano-liquid chromatography and mass spectrometry. The results obtained after simulated gastrointestinal digestion indicated that peptide fractions obtained after chromatographic separation significantly maintain their activity.

Isolating and Analyzing Protein Containing Granules from Cells

Recent advancements in detection methods have made protein condensates, also called granules, a major area of study, but tools to characterize these assemblies need continued development to keep up with evolving paradigms. We have optimized a protocol to separate condensates from cells using chemical cross-linking followed by size-exclusion chromatography (SEC). After SEC fractionation, the samples can be characterized by a variety of approaches including enzyme-linked immunosorbent assay, dynamic light scattering, electron microscopy, and mass spectrometry. The protocol described here has been optimized for cultured mammalian cells and E. coli expressing recombinant proteins. Since the lysates are fractionated by size, this protocol can be modified to study other large protein assemblies, including the nuclear pore complex, and for other tissues or organisms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: SEC separation of cross-linked mammalian cell lysates Alternate Protocol: Preparation of non-crosslinked mammalian cells Basic Protocol 2: SEC separation of E. coli lysate Support Protocol 1: Detecting protein of interest by ELISA Support Protocol 2: TCA precipitation of SEC fractions.

Size exclusion-reverse liquid column chromatography-mass spectrometry and its application in the identification of post-translationally modified proteins in rat kidney

LC-MS联用技术在蛋白质组学研究中具有重要的作用,但是在复杂的生物体系中,由于样品的高度复杂性和其中蛋白质含量的巨大差异,执行全面且无倾向的蛋白质组分析是一项挑战。因此,在液相色谱分离中采用基于不同原理的色谱分离方法来降低蛋白质样本的复杂度,并对微量蛋白质进行富集,对后续采用质谱方法进行信息的采集和深入分析至关重要。在这里我们开发了一种基于尺寸排阻色谱(SEC)与反相液相色谱(RPLC)结合的新方法来进行复杂体系蛋白质的分离和鉴定,特别是对于微量蛋白质的分析。首先使用SEC对蛋白质进行分离和富集,并酶解成多肽,再通过RPLC-MS联用的方法对酶解后的多肽进行分离和鉴定。结果显示使用上述方法可以有效降低蛋白质样本的复杂度,并有效提高微量蛋白质的鉴定能力,可从大鼠肾脏鉴定出23621个肽段及1345个蛋白质,比常规的二维强阳离子交换-反相液相色谱法(2D SCX-RPLC)鉴定到的肽段及蛋白质分别多出69%及27%。此外,该方法对肾脏翻译后修饰(PTM)蛋白质的鉴定显示出更多的优势,翻译后修饰的多肽鉴定率显著增加,特别是磷酸化肽段的鉴定效率可达到靶向富集策略的水平。在此展示的SEC-RPLC-MS可以更好地了解蛋白质翻译后修饰对肾脏的影响,最终将有助于增加我们对正常的生理性肾功能以及病理过程机制的理解。

Retention Mechanism of Branched Macromolecules in Size Exclusion Chromatography

The theory of Stockmayer in the modifications of Thurmond and Zimm has been used for the description of the size exclusion chromatography separation of randomly branched molecules with tetrafunctional branch points. It is assumed that free chain ends, created by the branching process, cause the molecules to be entrapped in the pores of the column packing with the time of their release given by the exponential law characteristic for the monomolecular reactions. Using this assumption, the anomalous elution behavior of such molecules can be modeled. With increasing elution volume, the average values of radius of gyration and, to a lesser degree, of molecular weight decrease and, after passing a minimum, again increase in the low-molecular weight region.

Off-line comprehensive size exclusion chromatography × reversed-phase liquid chromatography coupled to high resolution mass spectrometry for the analysis of lignocellulosic biomass products

Biochemical and thermochemical processes are two pathways to convert lignocellulosic biomass into fuels and chemicals. Both conversion types produce aqueous complex samples containing many oxygenated chemical functions over a wide range of masses. Nowadays, composition of these biomass products is still largely unknown, especially their nonvolatile part (300–1000 Da) mostly made of carbohydrates and their derivatives. In the present study, size exclusion chromatography (SEC) was investigated and applied on water soluble phase of a fast pyrolysis bio-oil (thermochemical conversion) and on aqueous phase of pretreated wheat straw (biochemical conversion). An optimization of mobile phase composition using model molecules was necessary to limit non-steric interactions and elute all chemical families. At the end, separation of carbohydrates, heterosides and aromatic species was performed. The chemical organization of SEC chromatograms was confirmed by coupling SEC with a Fourier transform-ion cyclotron resonance mass spectrometer (FT-ICR MS) using electrospray ionization (ESI) in the negative mode. On-line SEC-UV/FT-ICR MS hyphenation was a powerful tool to provide exact mass distribution of samples and get molecular formulae classed by chemical family. To go further, the complementarity of SEC with reversed-phase liquid chromatography (RPLC) was established with an off-line comprehensive 2D-LC analysis of the two samples. First, 140 fractions were collected physically from SEC separation for each sample, then each fraction was analyzed by RPLC hyphenated to an Ion Trap - Time of Flight mass spectrometer (SEC × RPLC-UV/IT-TOF MS) using ESI in both positive and negative modes. This comprehensive approach combining 2D-LC and high resolution mass spectrometry nearly doubled the number of peaks detected in comparison with 1D RPLC analysis and thus offered well resolved 2D contour plots, considered as relevant analytical fingerprints of the aqueous phase of biomass samples.

Abstract 2225: Exosomes in plasma for protein based cancer biomarkers: A clinical research tool using UHPLC-MS

Abstract Introduction: Exosomes have been used as a source of biomarkers in diagnostics for many diseases, including cancer, and are now being used as therapeutic delivery systems. To date, the most efficient method for isolating exosomes from serum or plasma samples has been to use a size exclusion, chromatography (SEC) based isolation. The conditions of the SEC separation can be modified to yield extracellular vesicles of different sizes and diagnostic value. Apoptotic bodies, 50-5000nm, microvesicles, 50-1000nm, and exosomes, 30-120nm, all have different diagnostic and targeted applications. Here, we show a routine and robust method for the isolation and monitoring of proteins in exosomes isolated from plasma samples. The methods and conditions used here are focused on rapidly providing a high yield of exosome vesicles in a monodisperse 40nm size range from a plasma sample. Materials and methods: K2EDTA plasma samples from donor disease and control groups were collected with IRB approval. All SEC columns and reagents for exosome isolation were obtained as a kit (NX Prenatal, Houston, TX). The 40nm exosome fraction from the column was lysed, the proteins reduced, alkylated and digested with trypsin; the subsequent peptides were analyzed by ultra-high performance liquid chromatography (Vanquish Horizon, ThermoFisher Scientific), tandem mass spectrometry (Q-Exactive, ThermoFisher Scientific), to quantify and identify greater than 500 proteins from exosomes, isolated from a single plasma sample, in a 1 hour method. Subsequent isolations of 40nm exosome fractions were labeled with a sulfo-NHS-biotin linker, followed by lysis and enrichment with streptavidin columns, and an identical LC-MS analysis to obtain quantitative analysis of the surface membrane protein contents on the exosomes. Results: Raw data was analyzed using the label-free quantitation module in Proteome Discoverer (Thermofisher Scientific). Peptide peak areas from three technical replicate runs were used to ascertain the reproducibility of the method across both normal and disease samples. Membrane protein extracts LC-MS data was further grouped by Protein Center (ThermoFisher Scientific) by cell type and tissue of origin. Conclusions: The methods shown here provide a robust and efficient platform for 40nm exosome isolation from human plasma samples, and quantitative analysis of the protein content thereof for the purposes of biomarker discovery, quantification and screening purposes. Citation Format: David A. Sarracino, Xiaolei Xie, Courtney E. Martel, Shen Luan, Kevin Rosenblatt. Exosomes in plasma for protein based cancer biomarkers: A clinical research tool using UHPLC-MS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2225.

Comprehensive two-dimensional liquid chromatography for the characterization of acrylate-modified hyaluronic acid.

Hyaluronic acid and its acrylate derivatives are important intermediates for various pharmaceutical, biomedical, and cosmetic applications due to their biocompatibility and viscoelasticity properties. However, these polymers are inherently difficult to characterize due to their significant heterogeneity regarding molar mass and chemical composition (degree of substitution, DS). The present study describes the development of a comprehensive online two-dimensional liquid chromatography (2D-LC) approach to characterize hyaluronic acid and its acrylate derivatives (DS ranging from 0.4 to 3.1) in terms of molar mass and degree of substitution. In the first dimension of the 2D-LC method, separation according to chemical composition/DS was achieved by using a stepwise solvent gradient and a reversed phase C8 column. Fractions from the first dimension were automatically transferred to the second dimension comprising size exclusion chromatographic separation of the fractions according to molar mass. It was found that the hyaluronic acid derivatives were broadly distributed with regard to both chemical composition and molar mass. Fractions with different degrees of substitution were identified, and their molar mass distributions were determined. The study proved that comprehensive 2D-LC is a powerful approach to reveal the complex nature of hyaluronic acid and its derivatives. Graphical abstract.

Peptide identification in alcalase hydrolysated pollen and comparison of its bioactivity with royal jelly

Peptides with a similar antioxidant and ACE-inhibitory activity of royal jelly (RJ) generated from Alcalase hydrolysated pollen (AHP) were predicted by Response Surface Methodology (RSM). The model equations were proposed according to the effects of time and enzyme concentration on the antioxidant and ACE-inhibitory activity. The optimum values for Alcalase concentration and hydrolysis time were 1.5% and 4 h, respectively. Later, AHP was prepared and deproteinised to be further analysed using size-exclusion chromatography (SEC). After SEC separation, fractions with the highest activity of ACE-inhibitory, DPPH radical scavenging and ferric-reducing power were purified by RP-HPLC. The highest ACE-inhibitory and DPPH scavenging activity of fractions was found 100% and 66.61%, respectively. The most active fractions were analysed by nano-liquid chromatography and mass spectrometry in tandem (nLC-MS/MS) and a total of 195 peptide sequences were identified. The origins of all peptides were herbal proteins and certain coincidences with previously described bioactive sequences were discussed.