Low Molecular Weight Range Research Articles

In-depth characterization of N-glycosylation and sialic acid content in fetal and adult fibrinogen

BackgroundFetal fibrinogen is a variant present in neonates. Blood products used in neonates are tailored for adults and do not seamlessly integrate into neonatal clots. Increased sialic acid content has been found in fetal fibrinogen compared with adult fibrinogen. However, the extent or location of sialic acids on fibrinogen remains unknown. ObjectivesTo investigate differences in glycosylation and sialic acid content between fetal and adult fibrinogen. MethodsGlycans were eluted from human cord blood-isolated fetal fibrinogen and commercially available adult fibrinogen using filter-aided N-linked glycan separation. A α, B β, and λ chains were isolated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in-gel enzymatic digestion was performed. Infrared matrix-assisted laser desorption electrospray ionization mass spectrometry was used for analysis. ResultsIn total, 39 and 22 glycans were detected in fetal and adult fibrinogen, respectively. Fetal fibrinogen glycans were most abundant in the lower molecular weight range <4 kDa. After isolating the Aα, Bβ, and λ chains, increased glycosylation and sialic acid content was found in fetal fibrinogen. Increased glycosylation was detected across all 3 chains, and increased sialic acid content was found in the Bβ chain. ConclusionSialylation in the Bβ chain of fetal fibrinogen supports previous findings showing more knob ‘B’ interactions occur in fetal fibrinogen than in adult fibrinogen during clot polymerization. This is also the first detection of glycosylation in the Aα chain of fibrinogen. By elucidating the fibrinogen N-linked glycome, this study found where sialic acid content differs the most between adult and fetal fibrinogen. This can ultimately be used to develop blood products that are neonatal-compatible.

Differential effects of purified low molecular weight Poly(I:C) in the maternal immune activation model depend on the laboratory environment

The Poly (I:C) (polyriboinosinic-polyribocytidilic acid) paradigm of maternal immune activation (MIA) is most widely used as experimental model for the evaluation of the effects of gestational infection on the brain and behavior of the progeny. We have previously reported significant batch-to-batch variability in the effects of Poly (I:C), purchased from the same supplier (Sigma–Aldrich), on maternal and fetal immune responses and found these differences to be dependent on the relative amount of synthetic double-stranded RNA fragments in the high versus low molecular weight (LMW) range contained in the compound. We here resorted to Poly (I:C) purified for LMW dsRNA fragments to establish a MIA paradigm with increased reproducibility and enhanced standardization in an effort to refine the MIA paradigm and characterize its effect on offspring behavior. We found that the parallel application of LMW Poly (I:C) in two different MIA-experienced laboratories (Vienna and Zurich) yielded differential outcomes in terms of maternal immune responses and behavioral phenotypes in the offspring generation. In both experimental sites, administration of LMW Poly (I:C) induced a significant sickness response and cytokine induction in the pregnant dam and fetal brains, while the expected deficit in sociability as one main behavioral outcome parameter in the MIA progeny, was only present in the Zurich, but not the Vienna cohort. We conclude that although using Poly (I:C) purified for a defined molecular weight range reduces batch-to-batch variability, it does not make the MIA model more reliable and robust. The differential response in behavioral phenotypes of the MIA offspring between the two laboratories illustrates the highly complex interaction between prenatal and postnatal milieus - including the laboratory environment - that determine offspring phenotypic outcomes after MIA. Consequently, establishing a new MIA protocol or implementing the MIA model firstly under new or changed environmental conditions must include the assessment of offspring behavior to ensure solid and reproducible experimental outcomes.

Effects of heat‐moisture treatment on the multiscale structure and digestibility of sweet potato starch

SummaryThe effects of water content on the multi‐scale structure and digestibility of sweet potato starch (SPS) during heat‐moisture treatment (HMT) were studied, and the relationship between water content, starch structure and starch digestibility was analysed. The results showed that the surface of SPS granules changed after HMT, mainly in the form of depression, rupture and adhesion, and the change was more obvious with the increase of water content, and the crystalline structure changed from the original A‐type to A + V‐type. The relative crystallinity and double helix content decreased, the thickness of semi‐crystalline layered structure increased, and the peak intensity decreased. The molecular weight of SPS after HMT tended to be distributed in the low molecular weight range of 4 × 104 g mol−1 &lt; Mw &lt;4 × 105 g mol−1. These structural changes promoted the rearrangement of the intermolecular structure of SPS, which made amylase shield the original action site when acting on SPS, and converted part of rapidly digestible starch (RDS) content into slow digestible starch (SDS) and resistant starch (RS) content, resulting in the increase of SDS and RS content. Especially in HMT‐25, the RS content of SPS was the highest, which was 28.60%. The experimental data can provide theoretical support for broadening the application of SPS.

Schistosoma mansoni egg-derived thioredoxin and Sm14 drive the development of IL-10 producing regulatory B cells.

During chronic schistosome infections, a complex regulatory network is induced to regulate the host immune system, in which IL-10-producing regulatory B (Breg) cells play a significant role. Schistosoma mansoni soluble egg antigens (SEA) are bound and internalized by B cells and induce both human and mouse IL-10 producing Breg cells. To identify Breg-inducing proteins in SEA, we fractionated SEA by size exclusion chromatography and found 6 fractions able to induce IL-10 production by B cells (out of 18) in the high, medium and low molecular weight (MW) range. The high MW fractions were rich in heavily glycosylated molecules, including multi-fucosylated proteins. Using SEA glycoproteins purified by affinity chromatography and synthetic glycans coupled to gold nanoparticles, we investigated the role of these glycan structures in inducing IL-10 production by B cells. Then, we performed proteomics analysis on active low MW fractions and identified a number of proteins with putative immunomodulatory properties, notably thioredoxin (SmTrx1) and the fatty acid binding protein Sm14. Subsequent splenic murine B cell stimulations and hock immunizations with recombinant SmTrx1 and Sm14 showed their ability to dose-dependently induce IL-10 production by B cells both in vitro and in vivo. Identification of unique Breg cells-inducing molecules may pave the way to innovative therapeutic strategies for inflammatory and auto-immune diseases.

Global profiling of the RNA and protein complexes of Escherichia coli by size exclusion chromatography followed by RNA sequencing and mass spectrometry (SEC-seq).

New methods for the global identification of RNA-protein interactions have led to greater recognition of the abundance and importance of RNA-binding proteins (RBPs) in bacteria. Here, we expand this tool kit by developing SEC-seq, a method based on a similar concept as the established Grad-seq approach. In Grad-seq, cellular RNA and protein complexes of a bacterium of interest are separated in a glycerol gradient, followed by high-throughput RNA-sequencing and mass spectrometry analyses of individual gradient fractions. New RNA-protein complexes are predicted based on the similarity of their elution profiles. In SEC-seq, we have replaced the glycerol gradient with separation by size exclusion chromatography, which shortens operation times and offers greater potential for automation. Applying SEC-seq to Escherichia coli, we find that the method provides a higher resolution than Grad-seq in the lower molecular weight range up to ~500 kDa. This is illustrated by the ability of SEC-seq to resolve two distinct, but similarly sized complexes of the global translational repressor CsrA with either of its antagonistic small RNAs, CsrB and CsrC. We also characterized changes in the SEC-seq profiles of the small RNA MicA upon deletion of its RNA chaperones Hfq and ProQ and investigated the redistribution of these two proteins upon RNase treatment. Overall, we demonstrate that SEC-seq is a tractable and reproducible method for the global profiling of bacterial RNA-protein complexes that offers the potential to discover yet-unrecognized associations between bacterial RNAs and proteins.

Ti3C2(OH)x-assisted LDI-TOF-MS for the rapid analysis of natural small molecules.

The inhomogeneous distribution of co-crystallized analytes and the traditional organic matrices as well as the intensive background interference in the low molecular weight range hinder the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in the analysis of small-molecular compounds. New two-dimensional material MXene (e.g., Ti3C2) exerts better hydrophilicity, homogeneity and repeatability, and higher laser desorption efficiency, as well as less background interference than traditional organic matrices and other nanomaterial matrices such as titanium oxide, graphene, and gold nanostructures. This study was aimed to design Ti3C2 matrix with abundant hydroxyls on its surface, enhance the stability of this hydroxyl-rich Ti3C2 (Ti3C2(OH)x), and evaluate the analytical performances of Ti3C2(OH)x-assisted laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF-MS) for small-molecular natural compounds in complex samples. The developed Ti3C2(OH)x showed the distinct advantages such as minimum background interference, high peak intensity (~105), high salt (0.6 M) and protein (0.5 mg/mL) tolerance, good repeatability (relative standard deviation<20%), and good stability after eight months of storage. Ti3C2(OH)x-assisted LDI-TOF-MS analysis could be used to rapidly identify Artemisia annua (a world-famous traditional Chinese medicine) and quantify the contents of the main chemical ingredients (oxymatrine (OXY) and matrine) of Compound Kushen Injection (CKI). Interestingly, the content of OXY in CKI could be accurately quantified by Ti3C2(OH)x-assisted LDI-TOF-MS, and there was a good linear relationship (R2 -0.9929), a low limit of detection (400 pg), and a low limit of quantification (600 pg) of OXY. Taken together, the rapid and accurate analysis of small-molecular natural compounds in complicated samples could be achieved by the Ti3C2(OH)x-assisted LDI-TOF-MS analysis.

Trifluoperazine (TFP)-mediated fluorescence imaging approach reveals a probable calmodulin (CaM)-independent calcium signaling accompanying differential protein phosphorylation in NaCl-stressed sunflower seedlings (Helianthus annuus L. var. KBSH44)

The impact of calcium (Ca2+) under abiotic stress is manifested by both calmodulin (CaM)-dependent and calmodulin-independent pathways of signaling mechanisms in plants. Present work demonstrates the impact of NaCl (120 mM) stress on Ca2+-calmodulin activity and its spatial distribution in roots of 2d old sunflower seedlings (Helianthus annuus). NaCl stress significantly increases [Ca2+]cyt in roots (69%) which corresponds with 8% increase in activated calmodulin (Ca2+-CaM) levels. Thus, NaCl stress induced Ca2+ signaling in roots is likely to majorly operate through CaM-independent routes. Trifluoperazine-mediated fluorescence imaging reveals differential distribution of Ca2+-CaM complex in the elongation and differentiation zone of seedling roots which indicate spatial regulation of Ca2+-CaM signaling operative in the presence of NaCl stress. NaCl stress-induced CaM-independent Ca2+ signaling is further accompanied by differential protein phoshorylation of cytosolic proteins in seedling roots and cotyledons. Phosphoproteins exhibiting qualitative differences in roots in response to salt-stress were of both low and high molecular weight range (76–17.5 kDa) unlike in cotyledons where only high molecular weight (>76 kDa) phosphoproteins exhibited major differential expressions in response to 120 mM NaCl stress. Among the various polypeptides in the fraction of OB membrane proteins two polypeptides of 48 and >76 kDa exhibited increased protein phosphorylation in the presence of NaCl stress. NaCl stress for 48 h induces differences in the expression of phosphoproteins in roots and cotyledons, suggesting the long distance signaling of Na+ stress from roots to cotyledons. Roots seem to exhibit much stringent regulation of phosphoproteins in comparison with cotyledons which have lesser qualitative differences. Thus, present work provides evidence on the modulation of Ca2+-CaM signaling accompanying differential protein phosphorylation in sunflower seedling roots and cotyledons under NaCl stress.

Characterization of somatic and metabolic antigens of Cysticercus cellulosae and determination of immunodominant proteins

The present study shows the characterization of four somatic antigens (Whole Cyst Antigen (WCA), Cystic Fluid Antigen (CFA), Scolex Antigen (SA) and Membrane-Body Antigen (MBA)) and one metabolic antigen (Excretory-Secretory Antigen (ESA)) prepared from Cysticercus cellulosae originated from a naturally infected pig. Immunodominat proteins were determined using hyper immune rabbit sera. The Sodium Dodecyl SulphatePolyacrylamide Gel Electrophoresis (SDS-PAGE) profile of the antigens revealed different numbers and patterns of protein bands in the range of 11.80 to 176.74 kDa. The electrophoretic pattern of ESA also revealed a number of closely placed indistinct bands in the range of 20-34 kDa.. The proteins in the lower molecular weight range (&lt; 30 kDa) in the case of WCA, CFA, ESA, and the medium molecular weight range (30-60 kDa) in the case of SA and MBA were observed to be immunodominant in the Enzyme-Linked Immunoelectrotransfer Blot (EITB) assay. These immunodominant proteins could be further investigated as potential diagnostic antigens in the serodiagnosis of cysticercosis in pigs, and neurocysticercosis in human patients.

Von Willebrand Factor Multimer Analysis by Low Resolution SDS-Agarose Gel Electrophoresis.

Von Willebrand factor (VWF) is a complex glycoprotein found in plasma, composed of disulfide-bond-linked multimers with apparent molecular weights between 500 kDa and 20,000 kDa. After release of VWF from storage granules, it is cleaved in flowing blood by the specific metalloproteinase ADAMTS13, resulting in a highly characteristic cleavage pattern and structure. As the structure of VWF multimers determines diagnosis of von Willebrand disease, which has different sub-types with different multimer- and cleavage patterns, VWF analysis is performed using low-resolution horizontal SDS-agarose gel electrophoresis. However, almost every laboratory uses a different protocol, and all experimental details are rarely, if at all, described. Therefore, the results from similar methods may be substantially different. Here, we present a detailed description of a validated VWF multimer method that we have developed. It has been successfully used for over more than 20 years in quality control of recombinant and plasma-derived VWF drug products, and in preclinical and clinical studies with VWF drug candidates. As most of the published methods, it enables visualization of VWF multimers separated by electrophoresis by immunostaining with a polyclonal anti-human VWF antibody followed by a secondary antibody coupled to alkaline phosphatase. VWF appears as a series of regularly spaced bands on the low and middle molecular weight range of the gel, with an unresolved zone in the high molecular weight (HMW) range, where ultra-large multimers are found. An example is shown below. This low-resolution agarose gel electrophoresis allows the determination of the number of VWF multimers with high reproducibility. Graphical abstract: Example of electrophoretic analysis of multimer structure of four batches of a recombinant VWF drug substance.

Experimental and Theoretical Studies of Carboxylic Polymers with Low Molecular Weight as Inhibitors for Calcium Carbonate Scale

Poly acrylic acid (PAA) and polyepoxysuccinic acid (PESA) were investigated as scale inhibitors. The static experiments certified that PAA was superior to PESA for the inhibition of calcium carbonate in the low molecular weight range. The X-ray diffraction patterns suggest that the effect of PAA on the calcite (1 0 4) and (1 1 0) crystal plane was more obvious. Scanning electron microscopy was used to study the surface morphology of the depositions, which indicated that the addition of scale inhibitors could disturb the normal growth of CaCO3 scale. The transmittance ratio of ferric oxide demonstrated that PAA had a better dispersion performance than PESA. The molecular dynamics simulation and quantum calculation were selected to theoretically explore the mechanism and structure of scale inhibitors, indicating that the interaction of PAA with (1 0 4) and (1 1 0) calcite crystal surfaces was stronger than PESA. In addition, the results indicated that the PAA with negative charge more easily adsorbed free Ca2+ in the aqueous phase. Based on these observations, PAA exhibited better scale inhibition and dispersion effects than PESA in the case of low molecular weight.

Characterization of a Complex Mixture of Immunomodulator Peptides Obtained from Autologous Urine.

A complex mixture of peptides plays a key role in the regulation of the immune system; different sources as raw materials mainly from animals and vegetables have been reported to provide these extracts. The batch-to-batch product consistency depends on in-process controls established. However, when an immunomodulator is a customized product obtained from the same volunteer who will receive the product to personalize the treatment, the criteria to establish the consistency between volunteers are different. In this sense, it is expected to have the same molecular weight range although the profile of peptide abundance is different. Here, we characterized the peptide profile of three extracts of an immunomodulator obtained from the urine of different volunteers suffering from three different diseases (i.e., allergic rhinitis, rheumatoid arthritis, and chronic rhinopharyngitis), using size exclusion chromatography (SEC) and mass spectrometry (MS). The peptides contained in the immunomodulators were stable after six months, stored in a refrigerator. Our results showed a chromatographic profile with the same range of low molecular weight (less than 17 kDa) in all analyzed samples by SEC; these results were also confirmed by MS showing an exact mass spectrum from 3 to 13 kDa. The fact that the peptide profiles were conserved during a six-month period at refrigeration conditions (2 to 8°C) maintaining the quality and stability of the immunomodulator supports the notion that it might be an alternative in the treatment of chronic hypersensibility disorders.

Short-Term Iodine Dynamics in Soil Solution.

Assessing the reactions of iodine (I) in soil is critical to evaluate radioiodine exposure and understand soil-to-crop transfer rates. Our mechanistic understanding has been constrained by method limitations in assessing the dynamic interactions of iodine between soil solution and soil solid phase over short periods (hours). We use microdialysis to passively extract soil solution spiked with radioiodine (129I- and 129IO3-) to monitor short-term (≤40 h) in situ fixation and speciation changes. We observed greater instantaneous adsorption of 129IO3- compared to 129I- in all soils and the complete reduction of 129IO3- to 129I- within 5 h of addition. Loss of 129I from solution was extremely rapid; the average half-lives of 129I- and 129IO3- in soil solution were 4.06 and 10.03 h, respectively. We detected the presence of soluble organically bound iodine (org-129I) with a low molecular weight (MW) range (0.5-5 kDa) in all soils and a slower (20-40 h) time-dependent formation of larger MW org-I compounds (12-18 kDa) in some samples. This study highlights the very short window of immediate availability in which I from rainfall or irrigation remains in soil solution and available to crops, thus presenting significant challenges to phytofortification strategies in soil-based production systems.

Characterization of pyrolytic lignin and insight into its formation mechanisms using novel techniques and DFT method

Real pyrolytic lignin (R-PLs) and pyrolytic products from model compounds (M-PL) were characterized by HPLC/Qtof-MS, GPC and 2D HSQC NMR to acquire molecular information. A huge amount of molecules were detected, analyzed and sorted into several categories according to their properties. Most compounds in R-PLs have complicated sidechains, with high molecular weight (MW) but moderate ring double bond equivalent (RDB), which might be derivatives of lignin fragments. The low MW fractions of R-PLs are considered as lignin simple monomers (LSM) and their derivatives, because the peaks of R-PLs and M-PL within low MW range of GPC are proximal. Molecules with medium MW but pretty high RDB, supposed to be coke/PAHs, were found more in M-PL than R-PLs, indicating that coke/PAHs primarily derive from LSM. Formation mechanisms of PL were proposed based on the differences between R-PLs and M-PL, which were supported by density functional theory (DFT) calculation. The results provide an in-depth understanding of pyrolytic lignin and inspire studies on high-value utilization of pyrolytic lignin.

High Yielding Acid-Catalysed Hydrolysis of Cellulosic Polysaccharides and Native Biomass into Low Molecular Weight Sugars in Mixed Ionic Liquid Systems.

Ionic media comprising 1‐butyl‐3‐methylimidazolium chloride and the acidic deep eutectic solvent choline chloride/oxalic acid as co‐solvent‐catalyst, very efficiently convert various cellulosic substrates, including native cellulosic biomass, into water‐soluble carbohydrates. The optimum reaction systems yield a narrow range of low molecular weight carbohydrates directly from cellulose, lignocellulose, or algal saccharides, in high yields and selectivities up to 98 %. Cellulose possesses significant potential as a renewable platform from which to generate large volumes of green replacements to many petrochemical products. Within this goal, the production of low molecular weight saccharides from cellulosic substances is the key to success. Native cellulose and lignocellulosic feedstocks are less accessible for such transformations and depolymerisation of polysaccharides remains a primary challenge to be overcome. In this study, we identify the catalytic activity associated with selected deep eutectic solvents that favours the hydrolysis of polysaccharides and develop reaction conditions to improve the outcomes of desirable low molecular weight sugars. We successfully apply the chemistry to raw bulk, non‐pretreated cellulosic substances.

Experimental Study of Thermal and Catalytic Pyrolysis of Plastic Waste Components

Thermal and catalytic pyrolysis of virgin low-density polyethylene (LDPE), high-density polyethylene (HDPE), polypropylene (PP) and mixtures of LDPE/PP were carried out in a 200 mL laboratory scale batch reactor at 460 °C in a nitrogen atmosphere. Thermogravimetric analysis (TGA) was carried out to study the thermal and catalytic degradation of the polymers at a heating rate of 10 °C/min. The amount of PP was varied in the LDPE/PP mixture to explore its effect on the reaction. In thermal degradation (TGA) of LDPE/PP blends, a lower decomposition temperature was observed for LDPE/PP mixtures compared to pure LDPE, indicating interaction between the two polymer types. In the presence of a catalyst (CAT-2), the degradation temperatures for the pure polymers were reduced. The TGA results were validated in a batch reactor using PP and LDPE, respectively. The result from thermal pyrolysis showed that the oil product contained significant amounts of hydrocarbons in the ranges of C7–C12 (gasoline range) and C13–C20 (diesel range). The catalyst enhanced cracking at lower temperatures and narrowed the hydrocarbon distribution in the oil towards the lower molecular weight range (C7–C12). The result suggests that the oil produced from catalytic pyrolysis of waste plastics has a potential as an alternative fuel.

PO-112 The exploration of constitutively expressed myokines utilizing tissue-engineered skeletal muscle

Objective Recent evidence has identified skeletal muscle as a secretory organ. Many myokines, which are bioactive substances secreted from skeletal muscle, have been identified in plane muscle culture cells. Compared to the plane muscle culture cells, the tissue-engineered muscle is an excellent model as culture system mimicked native skeletal muscle. However, constitutively expressed genes and secreted compounds from tissue-engineered muscle have not been analyzed sufficiently. The purposes of this study were 1) to clarify kinetics of constitutively secreted compounds, and 2) to explore constitutively expressed genes in the tissue-engineered muscle.&#x0D; Methods C2C12 cells embedded within collagen gel solution were placed between two tendons made up of elastase-treated acelluar porcine blood vessel. The constructs were cultured in growth media for 2 days and cultured in differentiation media for 6 days. To compare with plane culture cells, C2C12 cells were cultured in plane under the same condition as the construct. The culture media were obtained, and analyzed by MALDI-TOF Mass Spectrometry. Furthermore, constitutively up-regulated genes in tissue-engineered skeletal muscle were explored based on microarray analysis and confirmed by RT-PCR.&#x0D; Results MALDI-TOF Mass Spectrometry revealed that the number of detected peaks in tissue-engineered muscle was abundant compared to that of plane muscle culture cells, especially at range of low molecular weight. Furthermore, the detected peaks were substantially different among these culture media and specific peaks were identified in tissue-engineered muscle. Based on microarray analysis, the transcription of cholecystokinin identified, and confirmed the up-regulation in tissue-engineered skeletal muscle by RT-PCR.&#x0D; Conclusions These results suggested that the tissue-engineered muscle constitutively secreted many compounds compared to plane culture cells, especially at range of low molecular weight. Furthermore, the transcription of cholecystokinin was up-regulated in tissue-engineered skeletal muscle. Besides of the plane muscle culture cells, it is possible to expect to obtain novel myokines utilizing tissue-engineered muscle.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) proteomic profiling of cerebrospinal fluid in the diagnosis of enteroviral meningitis: a proof-of-principle study.

The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for diagnosing viral infections by directly testing clinical specimens has not previously been explored. In this proof-of-principle study, we tested the hypothesis that proteomic profiling of cerebrospinal fluid (CSF) by mass spectrometry may be useful in the diagnosis of enteroviral (EV) meningitis. A total of 114 cryopreserved CSF samples were analyzed, of which 47 were positive for EV and 67 were negative. Total CSF proteins were precipitated and subjected to MALDI-TOF-MS analysis in a low (2-20kDa) molecular weight range using a MicroFlex LT mass spectrometer. The whole data set was randomly split into a training set (n = 76 specimens) and a validation set (n = 38 samples). Backward/forward stepwise logistic regression analyses identified 30 peaks that were differentially present in EV-positive and EV-negative specimens. These were used to build a model which displayed an overall classification accuracy of 93%. The discriminative ability of the model was confirmed by using a validation sample set (overall accuracy 83%). In fact, the model was able to correctly classify 61 out of 67 EV-negative samples and 42 out of 47 EV-positive specimens. EV meningitis is associated with a distinctive protein profile that may be directly detectable in CSF specimens by MALDI-TOF-MS.

Uniform polysaccharide composite microspheres with controllable network by microporous membrane emulsification technique.

High resolution has been constantly pursued in both preparative and analytical chromatography. Chromatographic media are a key factor during the entire separation process. Tailor-made chromatographic media have gained more attention because of their adjustable structure appropriate for application. Uniform polysaccharide composite microspheres were prepared with a mixture of agarose and dextran solution by membrane emulsification technique for the first time. Their pore structure was deliberately regulated by adjusting boththe polysaccharide composition and the molecular weight of dextran. Compared with pure agarose microspheres, polysaccharide composite microspheres had a higher separation resolution and their separation range was controllable. By increasing agarose concentration and decreasing dextran concentration at the same time during the preparation of composite microspheres, the mean pore size increased first and then decreased later, and also the pore size distribution became narrower. By increasing the molecular weight of dextran, the pores became smaller with a narrower pore size distribution. Microspheres with a composition of 10% agarose/2% dextran T40 or 8% agarose/4% dextran T150 showed a higher separation resolution for proteins within range of low molecular weight. Furthermore, the mechanical strength of this composite microsphere was improved by adjusting its composition. Atomic force microscope (AFM) results showed that pores were distributed evenly on both the surface and the inner part of microspheres, beneficial for the passage of biomolecules. These novel uniform polysaccharide composite microspheres have great potential for high-resolution bioseparation. Graphical abstract ᅟ.

The Impact of Pre-Analytical Conditions on Human Serum Peptidome Profiling.

The successful use of proteomic technology for the discovery of clinically relevant, new candidate biomarkers, especially in the low molecular weight range (peptidome), calls for a careful consideration of standardized operating procedures (SOP) for pre-analytical variables, including samples handling and storage. The current lack of standardization, widely considered a relevant source of random and systematic errors, underlies the uncertainty of analytical results and poor comparability, especially in multi-centric or inter-laboratory studies. In their recent study, Tsuchida etal. used the MALDI-TOF/MS technique to investigate the effect of long-term storage at -20 °C, -80 °C, and in liquid nitrogen on serum samples obtained for peptidomic analyses. The authors have also evaluated the effects of different sample thawing modalities. By including results from the same series as that reported on in a previous publication, they have effectively defined some important requirements for the peptidomic analysis of serum samples (e.g., maximum time intervals between venepuncture and serum separation [1h], minimum temperature for long-term sera storage temperature [-80 °C], ideal conditions for sample thawing).

Enhancing reproducibility of SALDI MS detection by concentrating analytes within laser spot

Surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI TOF MS) has become one of the most important analytical methods due to its less interference at low molecular weight range. However, it is still a challenge to obtain a good reproducibility of SALDI TOF MS because of the inhomogeneous distribution of analyte molecules induced by coffee ring effect. We propose a universal and reliable method to eliminate the coffee ring effect by concentrating all the analyte molecules within the laser spot. This method exhibits an excellent reproducibility of spot-to-spot and substrate-to-substrate, and the relative standard deviations (RSDs) for different concentrations are lower than 12.6%. It also performs good linear dependency (R2 > 0.98) in the log-log plot with the concentration range of 1nM to 1μM, and the limit of detection for R6G is down to 1fmol.