Sequential Digestion Research Articles

Microencapsulation of Bifidobacterium breve to Enhance Microbial Cell Viability in Green Soybean Yogurt

Bifidobacteria, a major bacterial group, have several beneficial impacts on health, such as enhancing the intestinal flora by limiting the colonization of pathogenic microorganisms and stimulating the immune system. As a result, bifidobacteria have been extensively included in various food products. In this study, Bifidobacterium breve TISTR 2130 was microencapsulated using an emulsion technique with sodium alginate and calcium lactate in green soybean milk as wall materials. This study found that microbeads prepared with 2.0% (w/v) sodium alginate and 2.0% (w/v) calcium lactate had the highest microencapsulation efficiency (MEE) of 99.8% ± 0.07%. In addition, the viability of microencapsulated B. breve TISTR 2130 and free cells was evaluated following a simulated gastrointestinal treatment. Microencapsulated B. breve TISTR 2130 showed higher cell viability than free cells under the simulated gastrointestinal conditions. The viability reduction of free cells dropped substantially to zero after 1 h of incubation in simulated gastrointestinal juice (SIJ), while the viable cell count of microencapsulated B. breve TISTR 2130 remained greater than 5 log CFU/mL and the survival rate was greater than 64% at the end of the sequential digestion. During refrigerated storage of green soybean yogurt (GSY) fortified with microencapsulated B. breve, the viability of B. breve TISTR 2130, syneresis, and acidity decreased, while the pH and viscosity increased. Microencapsulated B. breve TISTR 2130 has the potential to be used as a probiotic fortification in GSY since the viability remained above the recommended minimal limit of 6 log CFU/mL for 10 days during refrigerated storage. The present study demonstrated that the optimized microencapsulated B. breve TISTR 2130 sodium alginate matrix could survive the human gastrointestinal tract to provide health benefits and the possibility of incorporation into functional foods.

Expression of GADD45G and CAPRIN1 in Human Nucleus Pulposus: Implications for Intervertebral Disc Degeneration.

Marked cellular changes occur in human intervertebral disc (IVD) degeneration during disc degeneration with biochemical changes. Genome-wide analysis of the DNA methylation profile has identified 220 differentially methylated loci associated with human IVD degeneration. Among these, two cell-cycle-associated genes, growth arrest and DNA damage 45 gamma (GADD45G) and cytoplasmic activation/proliferation-associated protein-1 (CAPRIN1), were focused on. The expression of GADD45G and CAPRIN1 in human IVDs remains unknown. We aimed to examine the expression of GADD45G and CAPRIN1 in human nucleus pulposus (NP) cells and evaluate those in human NP tissues in the early and advanced stages of degeneration according to Pfirrmann magnetic resonance imaging (MRI) and histological classifications. Human NP cells were cultured as monolayers after isolation from NP tissues by sequential enzyme digestion. Total RNA was isolated, and the mRNA expression of GADD45G and CAPRIN1 was quantified using real-time polymerase chain reaction. To examine the effects of pro-inflammatory cytokines on mRNA expression, human NP cells were cultured in the presence of IL-1β. Protein expression was evaluated using Western blotting and immunohistochemistry. GADD45G and CAPRIN1 expression was identified in human NP cells at both mRNA and protein levels. The percentage of cells immunopositive for GADD45G and CAPRIN1 significantly increased according to the Pfirrmann grade. A significant correlation between the histological degeneration score and the percentage of GADD45G-immunopositive cells was identified, but not with that of CAPRIN1-immunopositive cells. The expression of cell-cycle-associated proteins (GADD45G and CAPRIN1) was enhanced in human NP cells at an advanced stage of degeneration, suggesting that it may be regulated during the progression of IVD degeneration to maintain the integrity of human NP tissues by controlling cell proliferation and apoptosis under epigenetic alteration.

Integration of microalgae cultivation and anaerobic co-digestion with dairy wastewater to enhance bioenergy and biochemicals production

A sequential anaerobic digestion and phycoremediation process was employed to recover nutrients and remove pollutants from dairy wastewater (DW), while simultaneously producing biomethane and biochemicals. Anaerobic digestion of 100% DW achieved a methane content and production rate of 53.7% and 0.17 L/L/d, respectively. This was accompanied by the removal of 65.5% chemical oxygen demand (COD), 86% total solid (TS), and 92.8% volatile fatty acids (VFAs). The anaerobic digestate was then used to grow Chlorella sorokiniana SU-1. Using 25% diluted digestate as the medium, SU-1 could reach 4.64 g/L biomass concentration, with total nitrogen (TN), total phosphorus (TP) and COD removal efficiencies of 77.6%, 87.1% and 70.4%, respectively. The obtained microalgal biomass (contained 38.5% carbohydrates, 24.9% proteins, 8.8% lipids) was used to co-digest with DW, resulting in good methane production performance. Co-digestion with 25% (w/v) algal biomass obtained a higher CH4 content (65.2%) and production rate (0.16 L/L/d) than other ratios.

Effect of micronization on olive pomace biotransformation in the static model of colonic fermentation

The effect of granulometric fractionation and micronization of olive pomace (OP) on the biotransformation of phenolic compounds by intestinal microbiota was investigated in vitro. Three types of powdered OP samples were incubated with human feces to simulate colonic fermentation, after a sequential static digestion: non-fractionated OP (NF), granulometrically fractionated OP (GF) and granulometrically fractionated and micronized OP (GFM). GF and GFM favored the release of hydroxytyrosol, oleuropein aglycone, apigenin and phenolic acid metabolites in the first hours of colonic fermentation compared to NF (up to 41-fold higher). GFM caused higher release of hydroxytyrosol than GF. GFM was the only sample to release tyrosol and sustained tyrosol levels up to 24 h of fermentation. Micronization associated with granulometric fractionation was more efficient than granulometric fractionation alone to increase the release of phenolic compounds from the OP matrix during simulated colonic fermentation and can be further studied for nutraceutical purposes.

Novel method to specifically determine the structures of non-N297 glycans in IgGs.

Monoclonal antibodies comprise a major class of biologic therapeutics and are also extensively studied in immunology. Given the importance of glycans on antibodies, fluorescent labeling of enzymatically released glycans and their LC/MS analysis is routinely used for in-depth characterization of antibody glycosylation. In this technical note, we propose a method for facile characterization of glycans in the variable region of antibodies using sequential enzymatic digests with Endoglycosidase-S2 and RapidTM Peptide-N-Glycosidase-F followed by labeling with a fluorescent dye carrying an NHS-carbamate moiety. The results and proposed mechanism also suggest that the choice of glycosidases along with the labeling chemistry is critical for accurate glycan analysis for a desired application.

Effects of extrusion on structural properties, physicochemical properties and in vitro starch digestibility of Tartary buckwheat flour

Tartary buckwheat is a promising pseudocereal with health benefits. However, native Tartary buckwheat flour (NTBF) cannot satisfy the specific physicochemical properties required for food processing due to its gluten-free character. This study investigated structural and physicochemical properties, the starch digestibility of Tartary buckwheat flour pregelatinized with traditional extrusion processing technology (TEPT), and improved extrusion processing technology (IEPT). TEPT had a higher destructive effect on the starch structure than IEPT. Amorphous flakes and incomplete starch granules were observed in pregelatinized Tartary buckwheat flour (PTBF). After extrusion, the relative crystallinity, endothermic enthalpy, the slowly digested starch (SDS) and resistant starch content decreased by 23.76–70.44%, 48.91–100.00%, 28.43–88.65% and 53.23–78.79%, respectively. However, the amylose molecular weight of branched starches, the degree of gelatinization (DG), the water absorption index (WAI), the starch digestibility, the rapidly digested starch content, and the glycemic index increased by 29.00–119.04%, 26.05–1279.88%, 4.2–188.70%, 9.2–19.96%, 113.04–236.25%, and 25.55–37.00%, respectively. For IEPT, crystallinity decreased with the increase in extrusion temperature and the decrease in feed moisture, whereas the DG and WAI showed a contrary trend. Compared to TEPT (5.05%), PTBF obtained with IEPT retained a higher SDS content (11.28–31.85%). Starch digestion of all samples possessed a combination of sequential and parallel digestion patterns. Correlation analysis suggested that an increase in the destructive effect on the starch structure from IEPT to TEPT was responsible for the changes in physicochemical properties and improvement of in vitro starch digestibility. This study provides a scientific basis for the quality improvement in Tartary buckwheat starchy foods following extrusion.

Sonochemical effect on the degree of substitution of octenyl-succinic anhydride into waxy rice starch nanoparticles and study of gastro-intestinal hydrolysis using INFOGEST in vitro digestion method

Octenyl succinylation of starch nanoparticles has been proven to be effective in a variety of food industry applications such as fat replacement, thickening agents, emulsion formation, and delivery of bioactive compounds. In this study, waxy rice starch was debranched with pullulanase to obtain short glucans, which were then modified with octenyl succinic anhydride (OSA) to obtain amphiphilic short glucans (ASG) using high (400 W) and low (200 W) ultrasonic power intensity at 30 and 60 cycles. Developed ASG were characterized by nanoparticle size (ca. < 50 nm), surface hydrophobicity and gastro-intestinal stability. Ultrasonic intervention progressively increased the degree of substitution (DS) of OSA into hydrolysed starch. A significant molecular exchange between starch and OSA was confirmed with shoulder peak at 1.07 ppm by 1H NMR, and 1560 and 1727 cm−1 peaks in FTIR spectral image, and broad band at 1260 cm−1 by Raman spectroscopy. The XRD analysis confirmed the change in crystalline structure, and the emergence of new peaks at 2θ angles of around 5.81° which represent the development of B-type structure following pullulanasehydrolysis, and minor peaks at 13.92° and 19.83°, which imply the production of Vh type structures in ASG. Gastro-intestinal hydrolysis of starch after modification was analyzed in a sequential digestion process using INFOGEST method. The gastro-kinetic studies unveiled the reduction in the digestibility constant in the oral-gastric phase, with significantly enhanced value of kinetic constants in the intestinal phase, proving a sustained gastro-intestinal stability. The OSA-modified starches with greater DS havemore rigid and compact surface structure, which provides superior protection against biochemical conditions in the stomach fluid.

The role of site-2-proteases in bacteria: a review on physiology, virulence, and therapeutic potential.

Site-2-proteases are a class of intramembrane proteases involved in regulated intramembrane proteolysis. Regulated intramembrane proteolysis is a highly conserved signaling mechanism that commonly involves sequential digestion of an anti-sigma factor by a site-1- and site-2-protease in response to external stimuli, resulting in an adaptive transcriptional response. Variation of this signaling cascade continues to emerge as the role of site-2-proteases in bacteria continues to be explored. Site-2-proteases are highly conserved among bacteria and play a key role in multiple processes, including iron uptake, stress response, and pheromone production. Additionally, an increasing number of site-2-proteases have been found to play a pivotal role in the virulence properties of multiple human pathogens, such as alginate production in Pseudomonas aeruginosa, toxin production in Vibrio cholerae, resistance to lysozyme in enterococci and antimicrobials in several Bacillus spp, and cell-envelope lipid composition in Mycobacterium tuberculosis. The prominent role of site-2-proteases in bacterial pathogenicity highlights the potential of site-2-proteases as novel targets for therapeutic intervention. In this review, we summarize the role of site-2-proteases in bacterial physiology and virulence, as well as evaluate the therapeutic potential of site-2-proteases.

De novo glycan sequencing by electronic excitation dissociation MS2-guided MS3 analysis on an Omnitrap-Orbitrap hybrid instrument.

Comprehensive de novo glycan sequencing remains an elusive goal due to the structural diversity and complexity of glycans. Present strategies employing collision-induced dissociation (CID) and higher energy collisional dissociation (HCD)-based multi-stage tandem mass spectrometry (MSn) or MS/MS combined with sequential exoglycosidase digestions are inherently low-throughput and difficult to automate. Compared to CID and HCD, electron transfer dissociation (ETD) and electron capture dissociation (ECD) each generate more cross-ring cleavages informative about linkage positions, but electronic excitation dissociation (EED) exceeds the information content of all other methods and is also applicable to analysis of singly charged precursors. Although EED can provide extensive glycan structural information in a single stage of MS/MS, its performance has largely been limited to FTICR MS, and thus it has not been widely adopted by the glycoscience research community. Here, the effective performance of EED MS/MS was demonstrated on a hybrid Orbitrap-Omnitrap QE-HF instrument, with high sensitivity, fragmentation efficiency, and analysis speed. In addition, a novel EED MS2-guided MS3 approach was developed for detailed glycan structural analysis. Automated topology reconstruction from MS2 and MS3 spectra could be achieved with a modified GlycoDeNovo software. We showed that the topology and linkage configurations of the Man9GlcNAc2 glycan can be accurately determined from first principles based on one EED MS2 and two CID-EED MS3 analyses, without reliance on biological knowledge, a structure database or a spectral library. The presented approach holds great promise for autonomous, comprehensive and de novo glycan sequencing.

Endogenous protein and lipid facilitate the digestion process of starch in cooked quinoa flours

Quinoa, known for its nutritional quality and as a low GI food, has arose great interests of scientists in the recent past. Intrinsic properties of starch as well as endogenous proteins and lipids might be important factors influencing the digestion processes of cooked quinoas. This work aims to investigate the roles of endogenous proteins and lipids on starch digestibility in the quinoa flour, and uncover the physicochemical and textural properties related to the digestion process. Quinoa flours with/without proteins and/or lipids removal were obtained and then subjected to an in vitro digestion before/after cooking. Interestingly, the cooked whole quinoa flours had the highest starch digestibility and eGI, followed by the flours with protein or lipid removal, with the smallest digestibility found in the pure quinoa starch. This was in an opposite order of the equivalent raw quinoa flours, where proteins and lipids strongly inhibit the starch digestion. The cooked whole quinoa flours showed a first-order kinetic digestion pattern, with continuous hydrolysis of the long amylose chains, compared to combination of parallel and sequential digestion kinetic observed for other quinoa samples and the reduction in amylopectin with DP ∼20–250 noted during the fast digestion stage. Additionally, compared to whole quinoa flours, samples with protein and/or lipid removal displayed the higher final viscosity and shear-thinning property, with aggregated starch observed under confocal laser scanning microscopy. It suggested that gel network is a primary factor affecting the digestion process of starch in different quinoa flours. Ultimately, this work provides new insights for the development and processing of healthy quinoa products. • Whole quinoa flours had the highest starch digestibility and eGI. • The cooked whole quinoa flours showed a first-order kinetic digestion pattern. • Samples with protein and lipid removal displayed higher final viscosity. • Aggregated starch observed under confocal laser scanning microscopy. • Gel network is potentially a primary factor affecting the digestion process.

Biohythane: a Potential Biofuel of the Future.

Today, the world is becoming more dependent on fossil fuels. The major drawbacks of these non-renewable energy resources include an extreme environmental pollution and an extinction threat. Several technologies including microalgal biodiesel production, biomass gasification, and bioethanol production have been explored for the generation of renewable energy especially, biofuels. One such promising research has been carried out in the generation of biohythane which has the potential to become an alternative fuel to the existing non-renewable ones. It has been reported that biohydrogen can be produced from organic wastes or agricultural feedstocks with the help of acidogens. Dark fermentation can be carried out by acidogens to produce biohydrogen under anaerobic conditions by utilizing lignocellulosic biomass or sugarcane feedstocks in the absence of light. The spent medium contains volatile short-chain fatty acids like acetate, butyrate, and propionate that can serve as substrates for acetogenesis followed by methane biosynthesis by methanogens. Therefore, the sequential two-stage anaerobic digestion (AD) involves a production of biohydrogen followed by the biosynthesis of methane. This combined process is termed as a single eponym "Biohythane" (hydrogen + methane). Several studies have demonstrated about the effectiveness of biofuel, and it is believed to have a greater energy recovery, environmental friendliness, and shorter fermentation time. Biohythane can serve as an alternative future green biofuel and solve the present energy crisis in India as well as the entire world.

53Mn‐53Cr chronology and ε54Cr‐Δ17O genealogy of Erg Chech 002: The oldest andesite in the solar system

AbstractThe meteorite Erg Chech (EC) 002 is the oldest felsic igneous rock from the solar system analyzed to date and provides a unique opportunity to study the formation of felsic crusts on differentiated protoplanets immediately after metal–silicate equilibration or core formation. The extinct 53Mn‐53Cr chronometer provides chronological constraints on the formation of EC 002 by applying the isochron approach using chromite, metal–silicate–sulfide, and whole‐rock fractions as well as “leachates” obtained by sequential digestion of a bulk sample. Assuming a chondritic evolution of its parent body, a 53Cr/52Cr model age is also obtained from the chromite fraction. The 53Mn‐53Cr isochron age of 1.73 ± 0.96 Ma (anchored to D'Orbigny angrite) and the chromite model age constrained between and Ma after the formation of calcium‐aluminium‐rich inclusions (CAIs) agree with the 26Al‐26Mg ages (anchored to CAIs) reported in previous studies. This indicates rapid cooling of EC 002 that allowed near‐contemporaneous closure of multiple isotope systems. Additionally, excess in the neutron‐rich 54Cr (nucleosynthetic anomalies) combined with mass‐independent isotope variations of 17O provides genealogical constraints on the accretion region of the EC 002 parent body. The 54Cr and 17O isotope compositions of EC 002 confirm its origin in the “noncarbonaceous” reservoir and overlap with the vestoid material Northwest Africa 12217 and anomalous eucrite Elephant Moraine 92023. This indicates a common feeding zone during accretion in the protoplanetary disk between the source of EC 002 and vestoids. The enigmatic origin of iron meteorites remains still unresolved as EC 002, which is more like a differentiated crust, has an isotope composition that does not match known iron meteorite groups that were once planetesimal cores.

Sequential ultrasound-assisted digestion procedure for determination of cadmium and lead contaminants in sea grapes and some seaweed products

Sequential ultrasound-assisted digestion procedure for determination of cadmium and lead contaminants in sea grapes and some seaweed products

Disulfide bonds in the SAPA domain of the pulmonary surfactant protein B precursor

The disulfide bonds formed in the SAPA domain of a recombinant version of the NH2-terminal propeptide (SP-BN) from the precursor of human pulmonary surfactant protein B (SP-B) were identified through sequential digestion of SP-BN with GluC/trypsin or thermolysin/GluC, followed by mass spectrometry (MS) analysis. MS spectra allowed identification of disulfide bonds between Cys32-Cys49 and Cys40-Cys55, and we propose a disulfide connectivity pattern of 1–3 and 2–4 within the SAPA domain, with the Cys residues numbered according to their position from the N-terminus of the propeptide sequence. The peaks with m/z ∼ 2136 and ∼ 1780 in the MS spectrum of the GluC/trypsin digest were assigned to peptides 24AWTTSSLACAQGPE37 and 45QALQCR50 linked by Cys32-Cys49 and 38FWCQSLE44 and 51ALGHCLQE58 linked by Cys40-Cys55 respectively. Tandem mass spectrometry (MS/MS) analysis verified the position of the bonds. The results of the series ions, immonium ions and internal fragment ions were all compatible with the proposed 1–3/2–4 position of the disulfide bonds in the SAPA domain. This X-pattern differs from the kringle-type found in the SAPB domain of the SAPLIP proteins, where the first Cys in the sequence links to the last, the second to the penultimate and the third to the fourth one. Regarding the SAPB domain of the SP-BN propeptide, the MS analysis of both digests identified the bond Cys100-Cys112, numbered 7–8, which is coincident with the bond position in the kringle motif. SignificanceThe SAPLIP (saposin-like proteins) family encompasses several proteins with homology to saposins (sphingolipids activator proteins). These are proteins with mainly alpha-helical folds, compact packing including well conserved disulfide bonds and ability to interact with phospholipids and membranes. There are two types of saposin-like domains termed as Saposin A (SAPA) and Saposin B (SAPB) domains. While disulfide connectivity has been well established in several SAPB domains, the position of disulfide bonds in SAPA domains is still unknown. The present study approaches a detailed proteomic study to determine disulfide connectivity in the SAPA domain of the precursor of human pulmonary surfactant-associated protein SP-B. This task has been a challenge requiring the combination of different sequential proteolytic treatments followed by MS analysis including MALDI-TOF and tandem mass MS/MS spectrometry. The determination for first time of the position of disulfide bonds in SAPA domains is an important step to understand the structural determinants defining its biological functions.

Preparation and Antioxidant Activities of High Fischer's Ratio Oligopeptides from Goat Whey.

This study aimed to obtain high Fischer’s ratio oligopeptides from goat whey (HFO) and investigate antioxidant property of it. Hydrolysis of goat whey was done with the approach of sequential digestion of pepsin and flavourzyme. With the adsorption of aromatic amino acids by activated carbon, HFO with a Fischer’s ratio of 27.070 and a molecular weight of 200–1,000 Da were obtained, and the branched-chain amino acids accounted for 22.87%. Then the antioxidant activity of HFO was evaluated. At the concentrations of 2.0 mg/mL and 0.50 mg/mL, HFO scavenged 77.27% and 99.63% of 1,1-diphenyl-2-picrylhydrazyl and 3-ethylbenzthiazoline-6-sulphonate free radicals respectively. The scavenging rate of HFO against hydroxyl radicals reached 92.31% at the concentration of 0.25 mg/mL. Animal experiments demonstrated that HFO could moderate the changes of malondialdehyde, superoxide dismutase and glutathione peroxidase caused by CCl4-induced oxidative stress in vivo. This study indicated that HFO from goat whey was capable of oxidation resistance both in vivo and in vitro, which provided a scientific basis for the high-value processing and application of goat milk whey.

A novel method to purify small RNAs from human tissues for methylation analysis by LC-MS/MS.

Methylation modification of small RNAs, including miRNA, piRNA, and tsRNA, is critical for small RNA biogenesis and biological function. Methylation of individual small RNA can be defined by liquid chromatography-coupled with mass spectrometry (LC-MS/MS). However, LC-MS/MS analysis requires a high purity of individual small RNA. Due to the difficulty of purifying specific small RNA from tissues or cells, the progress in characterizing small RNA methylation by LC-MS/MS is limited. Here, we report a novel method that can efficiently purify small RNA from human tissues for LC-MS/MS analysis. This method includes two steps: 1) pull down the target small RNA by incubating total small RNAs (18–24 nt) extracted from human tissues with a biotinylated antisense oligonucleotide of the target small RNA, followed by capturing the binding duplex of biotinylated antisense and small RNA via streptavidin magnetic beads, and 2) protect the target small RNA by pairing it with a single-strand DNA, which sequence is complementary to the target small RNA, to form a DNA/RNA hybrid double-strand, followed by sequential digestion with exonuclease I, nuclease S1, and DNase I, respectively. Furthermore, employing a mixture of four pairs of synthetic methylated and non-methylated small RNAs, we further refined this two-step method by optimizing the nuclease S1 treatment condition. With this method, we successfully purified miR-21-5p, miR-26-5p, piR-020485, and tsRNA from human lung and sperm tissue samples and analyzed their 2′-O-methylation modification at the 3′-end by LC-MS/MS.

Icariin Exerts Estrogen-Like Actions on Proliferation of Osteoblasts in Vitro via Membrane Estrogen Receptors-Mediated Non-nuclear Effects.

According to reports, icariin (ICA) is a bone anabolic agent able to prevent osteoporosis in both ovariectomized rats and postmenopausal women. However, its effect on osteoblast proliferation remains to be determined, and the underlying mechanism remains to be elucidated. Icariin-bovine serum albumin (BSA) conjugates were purified by Sephadex G-25 gel chromatography technology. Primary osteoblasts from neonatal rats were used to evaluate the effects of ICA, ICA-BSA, ICA-BSA + ICI182780, and ICA-BSA + PD98059. 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and propidium iodide (PI)-staining assays were used to detect the proliferation of osteoblasts after drug exposure. The intracellular calcium ions were detected using a confocal microscope with Fluo-3/AM as the fluorescent indicator. Western blot was capitalized on to measure the relative content of phospho-extracellular signal-regulated kinase (p-ERK). Primary osteoblasts in culture were detected by histochemical staining of alkaline phosphatase, and calcified nodules were obtained by sequential digestion. Icariin and bovine serum albumin could form conjugate, which could be purified by Sephadex G-25 gel chromatography technology. MTT and flow cytometry results show that ICA-BSA conjugate significantly facilitated the proliferation of osteoblasts (P < 0.05). The intracellular calcium ions also ascended vastly in the cells treated with ICA-BSA conjugate (P < 0.01). Icariin-bovine serum albumin exposure rapidly activated the extracellular signal-regulated kinase (ERK) signaling. Furthermore, ICA- and ICA-BSA-mediated actions on osteoblasts were signally alleviated after dealing with ERK inhibitor PD98059 or estrogen receptor (ER) antagonist ICI182780, which might have a relation to the repression of ERK phosphorylation. Icariin could serve as estrogen in osteoblast cells by the rapid nongenomic ER signaling pathway independent of ligand and estrogen response element (ERE) and mediated by mitogen-activated protein kinase (MAPK).

Optimized Workflow for Enrichment and Identification of Biotinylated Peptides Using Tamavidin 2-REV for BioID and Cell Surface Proteomics.

Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the level of biotinylated proteins in cells is low; therefore, large amounts of cellular proteins were required to detect biotinylated peptides. In addition, the enriched biotinylated peptide solution contained many contaminant ions. Here, we optimized the workflow for efficient enrichment of biotinylated peptides and removal of contaminant ions. The efficient recovery of biotinylated peptides with fewer contaminant ions was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and using optimal temperature and salt concentration for elution. The optimized workflow enabled identification of nearly 4-fold more biotinylated peptides with higher purity from RAW264.7 macrophages expressing TurboID-fused STING (stimulator of interferon genes). In addition, sequential digestion with Glu-C and trypsin revealed biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon epidermal growth factor (EGF) stimulation. This workflow will be useful for BioID and cell surface proteomics and for various other applications based on protein biotinylation.

Impact of Food Matrices on Digestibility of Allergens and Poorly Allergenic Homologs.

BackgroundProtease resistance is considered a risk factor for allergenicity of proteins, although the correlation is low. It is nonetheless a part of the weight-of-evidence approach, proposed by Codex, for assessing the allergenicity risk of novel food proteins. Susceptibility of proteins to pepsin is commonly tested with purified protein in solution.ObjectiveFood proteins are rarely consumed in purified form. Our aim was to evaluate the impact of experimental and endogenous food matrices on protease susceptibility of homologous protein pairs with different degrees of allergenicity.MethodsPorcine and shrimp tropomyosin (ST) were subjected to sequential exposure to amylase, pepsin, and pancreatin in their respective endogenous matrix (pork tenderloin/boiled shrimp) and in three different experimental matrices (dessert mousse [DM], soy milk [SM], and chocolate bar [CB]). Digestion was monitored by immunoblotting using tropomyosin-specific antibodies. Recombinant peach and strawberry lipid transfer protein were biotinylated, spiked into both peach and strawberry fruit pulp, and subjected to the same sequential digestion protocol. Digestion was monitored by immunoblotting using streptavidin for detection.ResultsChocolate bar, and to a lesser extent SM, had a clear protective effect against pepsin digestion of porcine tropomyosin (PT) and to a lesser extent of ST. Increased resistance was associated with increased protein content. Spiking experiments with bovine serum albumin (BSA) confirmed the protective effect of a protein-rich matrix. The two tropomyosins were both highly resistant to pepsin in their protein-rich and lean native food matrix. Pancreatin digestion remained rapid and complete, independent of the matrix. The fat-rich environment did not transfer protection against pepsin digestion. Spiking of recombinant peach and strawberry lipid transfer proteins into peach and strawberry pulp did not reveal any differential protective effect that could explain differences in allergenicity of both fruits.ConclusionsProtein-rich food matrices delay pepsin digestion by saturating the protease. This effect is most apparent for proteins that are highly pepsin susceptible in solution. The inclusion of food matrices does not help in understanding why some proteins are strong primary sensitizers while homologs are very poor allergens. Although for induction of symptoms in food allergic patients (elicitation), a protein-rich food matrix that may contribute to increased risk, our results indicate that the inclusion of food matrices in the weight-of-evidence approach for estimating the potential risks of novel proteins to become allergens (sensitization), is most likely of very limited value.

Construction of an InstantPC-derivatized glycan glucose unit database: A foundation work for high-throughput and high-sensitivity glycomic analysis.

The glycosylation profile of biotherapeutic glycoproteins is a critical quality attribute that is routinely monitored to ensure desired product quality, safety and efficacy. Additionally, as one of the most prominent and complex post-translational modifications, glycosylation plays a key role in disease manifestation. Changes in glycosylation may serve as a specific and sensitive biomarker for disease diagnostics and prognostics. However, the conventional 2-aminobenzamide-based N-glycosylation analysis procedure is time-consuming and insensitive with poor reproducibility. We have evaluated an innovative streamlined 96-well-plate-based platform utilizing InstantPC label for high-throughput, high-sensitivity glycan profiling, which is user-friendly, robust and ready for automation. However, the limited availability of InstantPC-labeled glycan standards has significantly hampered the applicability and transferability of this platform for expedited glycan structural profiling. To address this challenge, we have constructed a detailed InstantPC-labeled glycan glucose unit (GU) database through analysis of human serum and a variety of other glycoproteins from various sources. Following preliminary hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection separation and analysis, glycoproteins with complex glycan profiles were subjected to further fractionation by weak anion exchange HILIC and exoglycosidase sequential digestion for cross-validation of the glycan assignment. Hydrophilic interaction ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry was subsequently utilized for glycan fragmentation and accurate glycan mass confirmation. The constructed InstantPC glycan GU database is accurate and robust. It is believed that this database will enhance the application of the developed platform for high-throughput, high-sensitivity glycan profiling and that it will eventually advance glycan-based biopharmaceutical production and disease biomarker discovery.