Phosphorylation and ubiquitination of OsWRKY31 are integral to OsMKK10-2-mediated defense responses in rice.

Mitogen-activated protein kinase (MPK) cascades play vital roles in plant innate immunity, growth, and development. Here, we report that the rice (Oryza sativa) transcription factor gene OsWRKY31 is a key component in a MPK signaling pathway involved in plant disease resistance in rice. We found that the activation of OsMKK10-2 enhances resistance against the rice blast pathogen Magnaporthe oryzae and suppresses growth through an increase in jasmonic acid and salicylic acid accumulation and a decrease of indole-3-acetic acid levels. Knockout of OsWRKY31 compromises the defense responses mediated by OsMKK10-2. OsMKK10-2 and OsWRKY31 physically interact, and OsWRKY31 is phosphorylated by OsMPK3, OsMPK4, and OsMPK6. Phosphomimetic OsWRKY31 has elevated DNA-binding activity and confers enhanced resistance to M. oryzae. In addition, OsWRKY31 stability is regulated by phosphorylation and ubiquitination via RING-finger E3 ubiquitin ligases interacting with WRKY 1 (OsREIW1). Taken together, our findings indicate that modification of OsWRKY31 by phosphorylation and ubiquitination functions in the OsMKK10-2-mediated defense signaling pathway.

11-Plex DiLeu Isobaric Labeling Enables Quantitative Assessment of Brain Region Protein Association Networks Impacted by the Gut Microbiome.

Gut microbiota can regulate host brain functions and influence various physiological and pathological processes through the brain-gut axis. To systematically elucidate the intervention of different gut environments on different brain regions, we implemented an integrated approach that combines 11-plex DiLeu isobaric tags with a "BRIDGE" normalization strategy to comparatively analyze the proteome of six brain regions in germ-free (GF)- and conventionally raised (ConvR)-mice. A total of 5945 proteins were identified and 5656 were quantifiable, while 1906 of them were significantly changed between GF- and ConvR-mice; 281 proteins were filtered with FC greater than 1.2 in at least one brain region, of which heatmap analysis showed clear protein profile disparities, both between brain regions and gut microbiome conditions. Gut microbiome impact is most overt in the hypothalamus and the least in the thalamus region. Collectively, this approach allows an in-depth investigation of the induced protein changes by multiple gut microbiome environments in a brain region-specific manner. This comprehensive proteomic work improves the understanding of the brain region protein association networks impacted by the gut microbiome and highlights the critical roles of the brain-gut axis.

A strategy for the enrichment and characterization of disulfide bond-contained proteins from Chinese cobra (Naja atra) venom.

A new strategy combined gold-coated magnetic nanocomposites assisted enrichment with mass spectrometry was developed for the characterization of disulfide bond-contained proteins from Chinese cobra (Naja atra) venom. In this work, core-shell nanocomposites were synthesized by the seed-mediated growth method and used for the enrichment of snake venom proteins containing disulfide bonds. A total of 3545 tryptic digested peptides derived from 96 venom proteins in Naja atra venom were identified. The venom proteins comprised 14 toxin families including three-finger toxins, phospholipase A2 , snake venom metalloproteinase, cobra venom factor, and so forth. Extra 16 venom proteins were detected exclusively in the nanocomposites set, among which 11 venom proteins were from the three-finger toxins family. In the present study, the proposed simple and efficient protocol replaced the tedious and laborious technologies commonly used for pre-separating crude snake venom, suggesting widely implementation in low-abundance or trace disulfide bond-contained proteins or peptides characterization.

Preparation of Antibacterial Gelatin/Genipin Nanofibrous Membrane for Tympanic Membrane Repair.

Tympanic membrane perforation (TMP), a common disease, often needs a scaffold as the patch to support surgery. Due to the environment of auditory meatus, the patch can be infected by bacteria that results in failure; therefore, the ideal scaffold may combine biomimetic and antibacterial features. In this work, gelatin was used as the electrospinning framework, genipin as the crosslinking agent, and levofloxacin as an antibacterial in order to prepare the scaffold for TMP. Different contents of levofloxacin have been added to gelatin/genipin. It was found that, with the addition of levofloxacin, the gelatin/genipin membranes exhibit improved hydrophilia and enhanced tensile strength. The antibacterial and cell-cultured experiments showed that the prepared antibacterial membranes had excellent antibacterial properties and good biocompatibility, respectively. In summary, levofloxacin is a good group for the gelatin/genipin scaffold because it improves the physical properties and antibacterial action. Compared with different amounts of levofloxacin, a gelatin/genipin membrane with 1% levofloxacin is more suitable for a TM.

Integrated Label-Free and 10-Plex DiLeu Isobaric Tag Quantitative Methods for Profiling Changes in the Mouse Hypothalamic Neuropeptidome and Proteome: Assessment of the Impact of the Gut Microbiome.

Gut microbiota can regulate host physiological and pathological status through gut-brain communications or pathways. However, the impact of the gut microbiome on neuropeptides and proteins involved in regulating brain functions and behaviors is still not clearly understood. To address the problem, integrated label-free and 10-plex DiLeu isobaric tag-based quantitative methods were implemented to compare the profiling of neuropeptides and proteins in the hypothalamus of germ-free (GF)- vs conventionally raised (ConvR)-mice. A total of 2943 endogenous peptides from 63 neuropeptide precursors and 3971 proteins in the mouse hypothalamus were identified. Among these 368 significantly changed peptides (fold changes over 1.5 and a p-value of <0.05), 73.6% of the peptides showed higher levels in GF-mice than in ConvR-mice, and 26.4% of the peptides had higher levels in ConvR-mice than in GF-mice. These peptides were mainly from secretogranin-2, phosphatidylethanolamine-binding protein-1, ProSAAS, and proenkephalin-A. A quantitative proteomic analysis employing DiLeu isobaric tags revealed that 282 proteins were significantly up- or down-regulated (fold changes over 1.2 and a p-value of <0.05) among the 3277 quantified proteins. These neuropeptides and proteins were mainly involved in regulating behaviors, transmitter release, signaling pathways, and synapses. Interestingly, pathways including long-term potentiation, long-term depression, and circadian entrainment were involved. In the present study, a combined label-free and 10-plex DiLeu-based quantitative method enabled a comprehensive profiling of gut microbiome-induced dynamic changes of neuropeptides and proteins in the hypothalamus, suggesting that the gut microbiome might mediate a range of behavioral changes, brain development, and learning and memory through these neuropeptides and proteins.

Preparation of a DNA-Tb-MOF conjugate as a time-resolved probe for the detection of SO2 derivatives through an off-on effect.

Herein we synthesize a DNA-sensitized Tb-MOF conjugate (DNA-Tb-MOF) as a time-resolved luminescent probe to sensitively and selectively assay SO2 and their derivatives (i.e., HSO3-) through a photoluminescence off-on effect. The charge and energy transfer mechanism enables the demonstration of the effect of the photoluminescence turn-on which results from the reaction between the amino group of the DNA-Tb-MOF conjugate and SO2/HSO3-. The results demonstrate that the DNA-Tb-MOF conjugate probe can sense SO2 and their derivatives (i.e., HSO3-) with a detection limit of 0.02 ppm. Moreover, the photoluminescence off-on effect can be observed even by the naked eye.

Simultaneous determination of sulfur compounds from the sulfur pathway in rat plasma by liquid chromatography tandem mass spectrometry: application to the study of the effect of Shao Fu Zhu Yu decoction.

A sensitive, accurate, and time-saving approach was developed for the simultaneous quantification of eight sulfur compounds in the sulfur pathway, which could reflect the status of an organism, including oxidative stress, signal transduction, enzyme reaction, and so on. In order to overcome the instability of highly reactive sulfhydryl compounds, N-ethylmaleimide derivatization was adopted to effectively protect sulfhydryl-containing samples. Using isotope-labeled glutathione (GSH-13C2, 15N), the validated method was demonstrated to offer satisfactory linearity, accuracy, and precision. Separation was done by UHPLC, using a BEH amide column. Accordingly, 0.1% formic acid acetonitrile was selected as the precipitant. A tandem mass spectrometer was coupled to the chromatographic system and afforded a detection limit of 0.2 ng/mL. Good linearity was maintained over a wide concentration range (r2 > 0.994), and the accuracy was in the range of 86.6-114% for all the studied compounds. The precision, expressed in RSD%, ranged from 1.1% to 9.4% as intraday variability and less than 13% as interday precision for all of the analytes. The approach was applied to study the potential therapeutic mechanism of a well-known traditional Chinese medicine, Shao Fu Zhu Yu decoction. The results suggested that Shao Fu Zhu Yu decoction might protect against oxidative damage by increasing the concentrations of sulfhydryl compounds. Graphical abstract An approach to quantitatively determining sulfur compounds in the sulfur pathway simultaneously wasestablished and applied to the study of the effect of Shao Fu Zhu Yu decoction.

Determination of the n-octanol/water partition coefficients of weakly ionizable basic compounds by reversed-phase high-performance liquid chromatography with neutral model compounds.

A strategy to utilize neutral model compounds for lipophilicity measurement of ionizable basic compounds by reversed-phase high-performance liquid chromatography is proposed in this paper. The applicability of the novel protocol was justified by theoretical derivation. Meanwhile, the linear relationships between logarithm of apparent n-octanol/water partition coefficients (logKow '') and logarithm of retention factors corresponding to the 100% aqueous fraction of mobile phase (logkw ) were established for a basic training set, a neutral training set and a mixed training set of these two. As proved in theory, the good linearity and external validation results indicated that the logKow ''-logkw relationships obtained from a neutral model training set were always reliable regardless of mobile phase pH. Afterwards, the above relationships were adopted to determine the logKow of harmaline, a weakly dissociable alkaloid. As far as we know, this is the first report on experimental logKow data for harmaline (logKow = 2.28 ± 0.08). Introducing neutral compounds into a basic model training set or using neutral model compounds alone is recommended to measure the lipophilicity of weakly ionizable basic compounds especially those with high hydrophobicity for the advantages of more suitable model compound choices and convenient mobile phase pH control.

[Regulation of ursolic acid on TNF-alpha and collagen in silicotic rats].

OBJECTIVE: To explore the regulation of ursolic acid on the expressions of TNF-alpha and collagen on Silicotic Rats. METHODS: Seventy-five Wistar rats (class SPF) were divided into there groups according to the randomized block design, namely, control, model, ursolic acid groups with twenty-five rats in each group. SiO2 powders (250 mg/kg) were douched in the trachea of rat to make the silicotic model in model and ursolic acid groups. Ursolic acid (40 mg/kg) was injected into stomach cavity in ursolic acid group from the second day of SiO2, while the rats in control group were given sodium chloride in the same condition for 28 consecutive days. All rats were put to death on the 7th,14th and 28th day. TNF-alpha contents in serum were detected by ELISA. Total collagen contents in lung tissue were determined by hydroxyproline kits. Collagen I and III in lung tissue were investigated by western blot technique. RESULTS: After four weeks of intervention, the contents of TNF-alpha in serum of the model group had rised, showing statistically significant difference in each time compared to those of the control group (P < 0.05). Ursolic acid had depressant effect on the contents of TNF-alpha (P < 0.05). Compared with control group, the content of total collagen was significantly improved in model group (P < 0.05). However, ursolic acid depressed the expression of total collagen protein compared to those of the model group (P < 0.05). The expression levels of collagen I and III in ursolic acid and model groups were similar with the total collagen. CONCLUSIONS: Ursolic acid could decrease the expressions of TNF-alpha and collagen in the process of silicosis fibrosis.

Quantitative proteomic analysis reveals hub proteins for high temperature-induced male sterility in bread wheat (Triticum aestivum L.).

High-temperature (HT) stress can induce male sterility in wheat; however, the underlying mechanisms remain poorly understood. This study examined proteomic alterations across three developmental stages between normal and HT-induced male-sterile (HT-ms) anthers in wheat. Utilizing tandem mass tags-based proteomics, we identified 2532 differentially abundant proteins (DAPs): 27 in the tetrad stage, 157 in the binuclear stage, and 2348 in the trinuclear stage. Analyses through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways indicated significant enrichment of these DAPs in seven pathways, namely phenylpropanoid biosynthesis, flavonoid biosynthesis, sphingolipid metabolism, MAPK signaling pathway, starch and sucrose metabolism, response to heat, and response to reactive oxygen species (ROS). Our results indicated the downregulation of DAPs associated with phenylpropanoid biosynthesis and starch and sucrose metabolism, which aligns with anther indehiscence and the lack of starch in HT-ms anthers. By contrast, DAPs in the ROS pathway were upregulated, which aligns with excessive ROS accumulation in HT-ms anthers. Additionally, we conducted protein-protein interaction analysis for the DAPs of these pathways, identifying 15 hub DAPs. The abundance of these hub proteins was confirmed through qRT-PCR, assessing mRNA expression levels of the corresponding transcripts. Collectively, these results offer insights into the molecular mechanisms underlying HT-induced male sterility in wheat at the proteomic level, providing a valuable resource for further research in plant sexual reproduction.

Sex differences in diabetes­induced hepatic and renal damage.

Diabetes mellitus (DM) is a disease that affects millions of individuals worldwide and is characterized by abnormal glucose metabolism that can induce severe damage to numerous organs throughout the body. Sex differences have been demonstrated in a number of factors associated with diabetes and its complications, such as diabetic kidney disease and diabetic liver disease. To investigate the sex differences in DM further, the changes in the weight, food and water intake, and blood sugar of mice were recorded for 8 weeks in the present study. Hematoxylin and eosin staining, Masson's trichrome staining and transmission electron microscopy were used to observe the pathological changes of liver and kidney tissues. There is no significant difference in the water intake and blood glucose concentration between db/db female and male mice was observed. However, sex differences in liver and kidney damage including glomerular injury and hepatic fibrosis were found. In conclusion, the present study characterized the features of liver and kidney damage in db/db mice and indicated that sex differences should be taken into account in experiments using female and male experimental animals. Furthermore, sex differences should be taken into account in the selection of drug interventions in experiments and in clinical drug therapy.

Carp scales derived double cross-linking hydrogels achieve collagen peptides sustained-released for bone regeneration.

Collagen peptide exhibits a great activity in osteogenic differentiation and wound healing. However, uncontrolled collagen peptide release in bone defects leads to unsatisfactory bone regeneration. In this work, we prepared collagen peptide loaded calcium alginate hydrogel (SA-CP/Ca) derived from Asia carp scales by mixing sodium alginate solution, collagen peptides, calcium carbonate, covalent cross-linking agents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) in one pot. Physically and chemically double cross-linking realized higher crosslink density, smaller porosity and pore size, and higher energy storage modulus and loss modulus, achieving sustained release of collagen peptides. The release profile is fitted to Keppas-Sahlin model, to find SA-CP/Ca hydrogels are more inclined to release collagen peptides through expansion and degradation. The compatibility and osteogenic ability of SA-CP/Ca are demonstrated in vitro and in vivo.

HIF-1α participates in the regulation of S100A16-HRD1-GSK3ß/CK1α pathway in renal hypoxia injury.

S100 calcium-binding protein 16 (S100A16) is implicated in both chronic kidney disease (CKD) and acute kidney injury (AKI). Previous research has shown that S100A16 contributes to AKI by facilitating the ubiquitylation and degradation of glycogen synthase kinase 3ß (GSK3ß) and casein kinase 1α (CK1α) through the activation of HMG-CoA reductase degradation protein 1 (HRD1). However, the mechanisms governing S100A16-induced HRD1 activation and the upregulation of S100A16 expression in renal injury are not fully understood. In this study, we observed elevated expression of Hypoxia-inducible Factor 1-alpha (HIF-1α) in the kidneys of mice subjected to ischemia-reperfusion injury (IRI). S100A16 deletion attenuated the increased HIF-1α expression induced by IRI. Using a S100A16 knockout rat renal tubular epithelial cell line (NRK-52E cells), we found that S100A16 knockout effectively mitigated apoptosis during hypoxic reoxygenation (H/R) and cell injury induced by TGF-ß1. Our results revealed that H/R injuries increased both protein and mRNA levels of HIF-1α and HRD1 in renal tubular cells. S100A16 knockout reversed the expressions of HIF-1α and HRD1 under H/R conditions. Conversely, S100A16 overexpression in NRK-52E cells elevated HIF-1α and HRD1 levels. HIF-1α overexpression increased HRD1 and ß-catenin while decreasing GSK-3ß. HIF-1α inhibition restored HRD1 and ß-catenin upregulation and GSK-3ß downregulation by cellular H/R injury. Notably, Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated HIF-1α binding signals on the HRD1 promoter, and luciferase reporter gene assays confirmed HIF-1α's transcriptional regulation of HRD1. Additionally, we identified Transcription Factor AP-2 Beta (TFAP2B) as the upregulator of S100A16. ChIP and luciferase reporter assays confirmed TFAP2B as a transcription factor for S100A16. In summary, this study identifies TFAP2B as the transcription factor for S100A16 and demonstrates HIF-1α regulation of HRD1 transcription within the S100A16-HRD1-GSK3ß/CK1α pathway during renal hypoxia injury. These findings provide crucial insights into the molecular mechanisms of kidney injury, offering potential avenues for therapeutic intervention.

Identification and characterization of the CRK gene family in the wheat genome and analysis of their expression profile in response to high temperature-induced male sterility.

Cysteine-rich receptor-like kinases (CRKs) play many important roles during plant development, including defense responses under both biotic and abiotic stress, reactive oxygen species (ROS) homeostasis, callose deposition and programmed cell death (PCD). However, there are few studies on the involvement of the CRK family in male sterility due to heat stress in wheat (Triticum aestivum L.). In this study, a genome-wide characterization of the CRK family was performed to investigate the structural and functional attributes of the wheat CRKs in anther sterility caused by heat stress. A total of 95 CRK genes were unevenly distributed on 18 chromosomes, with the most genes distributed on chromosome 2B. Paralogous homologous genes with Ka/Ks ratios less than 1 may have undergone strong purifying selection during evolution and are more functionally conserved. The collinearity analysis results of CRK genes showed that wheat and Arabidopsis (A. thaliana), foxtail millet, Brachypodium distachyon (B. distachyon), and rice have three, 12, 15, and 11 pairs of orthologous genes, respectively. In addition, the results of the network interactions of genes and miRNAs showed that five miRNAs were in the hub of the interactions map, namely tae-miR9657b-5p, tae-miR9780, tae-miR9676-5p, tae-miR164, and tae-miR531. Furthermore, qRT-PCR validation of the six TaCRK genes showed that they play key roles in the development of the mononuclear stage anthers, as all six genes were expressed at highly significant levels in heat-stressed male sterile mononuclear stage anthers compared to normal anthers. We hypothesized that the TaCRK gene is significant in the process of high-temperature-induced sterility in wheat based on the combination of anther phenotypes, paraffin sections, and qRT-PCR data. These results improve our understanding of their relationship.

An experimental study of buckwheat polysaccharide in adjuvant therapy for S180 sarcoma mice.

Introduction: To discuss the auxiliary therapeutic effect of buckwheat polysaccharide (BP) on S180 sarcoma. Material and methods: Buckwheat polysaccharide was extracted with water and precipitated with ethanol. Solid tumor and ascites tumor mice models were established. The mice in the high, medium and low dosing groups (n = 24, each group) had their stomachs filled with different doses of BP. The cyclophosphamide (CTX) group and the model group (n = 24, each group) were used as control groups. The influence on the life cycle, the rate of suppressing the tumor, the thymus index, and the spleen index were evaluated. Tumor cells were cultured in vitro and intervened with drugs; flow cytometry was used to detect the changes in the cell cycle. Results: Buckwheat polysaccharide significantly improved the lifespan and survival rate of the mice. The group of mice treated with the medium dose showed the best survival rate when compared to the ones that received high and low doses of BP (p < 0.01). The tumor cells cultured in vitro were arrested in the G0/G1 phase to some extent (p > 0.05). The cyclophosphamide arrested the cycle of the tumor cells in the G2/M period (p < 0.01). Buckwheat polysaccharide could increase the thymus index, spleen index and the rate of suppressing the tumor, but the differences were not statistically significant. Conclusions: Buckwheat polysaccharide had no obvious effect in inhibiting the growth of tumors, but it significantly extended the lifespan, increased the survival rate and reduced the toxic effect of CTX.

Identification and application of species-specific peptide biomarkers from soft-shelled turtles (Pelodiscus sinensis) using post-translational modification detection-based peptidomics analysis.

The soft-shelled turtle is a commercially aquatic species in Asian countries, which serves as an important source of collagen with high nutritional and medicinal value, so it is of great significance to distinguish soft-shelled turtle derived collagen from others or adulterations. In this work, peptidomics analysis based on post-translational modification (PTM) assay was used to discover specific peptide biomarkers of soft-shelled turtle gelatin (STG). In total eight specific sequences and 74 peptides with different PTM types were screened out, and seven peptides with good signal responses and STG specificity were selected and validated as STG-specific peptide biomarkers. These peptide biomarkers could be used for distinguishing STG from other animal gelatins, and applied for ensuring the quality of collagens or gelatins from soft-shelled turtle with authenticity and traceability.

The effect of D-chiro-inositol on renal protection in diabetic mice.

D-Chiro-inositol (DCI) exerts a hypoglycaemic effect, participates in lipid metabolism and reduces kidney damage. In this study, we preliminarily explored the protective effect of DCI on renal injury in diabetic mice. Male db/db mice were used in this study. After treatment with DCI (35 and 70 mg/kg/d) for 6 consecutive weeks, random blood glucose (RBG) measurements were conducted at 0 and 6 weeks. Creatinine (Cr) and serum blood urea nitrogen (BUN) levels were measured using assay kit, and morphological changes in the kidneys were observed by HE staining, Masson staining and electron microscopy. Immunohistochemical and Western blot experiments were used to examine the protein expression of matrix metalloproteinase-9 (MMP-9), nuclear factor-κB (NF-κB) and peroxisome proliferator-activated receptor-γ (PPAR-γ). We discovered that the increased RBG levels were alleviated after treatment with DCI. Moreover, the Cr and BUN levels were reduced, glomerular mesangial hyperplasia was alleviated, and the degree of renal fibrosis was reduced. In addition, DCI improved the protein expression of MMP-9 and PPAR-γ in kidney tissue, which in turn inhibited NF-κB protein expression, as shown by immunohistochemistry and Western blotting. Our findings showed that DCI ameliorated the renal injury induced by diabetes by upregulating MMP-9 and PPAR-γ expression and downregulating NF-κB expression. We preliminarily concluded that the renal protective effect of DCI on diabetic mice may occurs through the MMP-9/NF-κB signalling pathway.

OsWRKY62 and OsWRKY76 Interact with Importin α1s for Negative Regulation of Defensive Responses in Rice Nucleus.

Background OsWRKY62 and OsWRKY76, two close members of WRKY transcription factors, function together as transcriptional repressors. OsWRKY62 is predominantly localized in the cytosol. What are the regulatory factors for OsWRKY62 nuclear translocation? Results In this study, we characterized the interaction of OsWRKY62 and OsWRKY76 with rice importin, OsIMα1a and OsIMα1b, for nuclear translocation. Chimeric OsWRKY62.1-GFP, which is predominantly localized in the cytoplasm, was translocated to the nucleus of Nicotiana benthamiana leaf cells in the presence of OsIMα1a or OsIMαΔIBB1a lacking the auto-inhibitory importin ß-binding domain. OsIMαΔIBB1a interacted with the WRKY domain of OsWRKY62.1, which has specific bipartite positively charged concatenated amino acids functioning as a nuclear localization signal (NLS). Similarly, we found that OsIMαΔIBB1a interacted with the AvrPib effector of rice blast fungus Magnaporthe oryzae, which contains a scattered distribution of positively charged amino acids. Furthermore, we identified a nuclear export signal (NES) in OsWRKY62.1 that inhibited nuclear transportation. Overexpression of OsIMα1a or OsIMα1b enhanced resistance to M. oryzae, whereas knockout mutants decreased resistance to the pathogen. However, overexpressing both OsIMα1a and OsWRKY62.1 were slightly more susceptible to M. oryzae than OsWRKY62.1 alone. Ectopic overexpression of OsWRKY62.1-NES fused gene compromised the enhanced susceptibility of OsWRKY62.1 to M. oryzae. Conclusion These results revealed the existence of NLS and NES in OsWRKY62. OsWRKY62, OsWRKY76, and AvrPib effector translocate to nucleus in association with importin α1s through new types of nuclear localization signals for negatively regulating defense responses.

Response boosting-based approach for absolute quantification of gelatin peptides using LC-MS/MS.

Response-boosting of MS signal was observed in gelatin samples due to abundant Glycine residues produced by collagen enzymolysis. In this work, a new strategy utilizing response-boosting to enhance detection sensitivity was developed for absolute quantification of Asini Corii Colla, a kind of gelatin commonly used as food therapy products in Asia, by high performance liquid chromatography coupled to tandem mass spectrometry. Peptidomics analysis was used to evaluate the similarity between eight different protein matrices, and deer-hide gelatin was selected as the appropriate simulated matrix. Isotope-labelled internal standard was used to compensate the matrix effect and construct matrix-matched calibration curves. The established method showed reliability in absolute quantification of three species-specific gelatin peptides with good linearity (r2 > 0.997), precision (RSD < 8.5%), repeatability (RSD < 8.9%), accuracy (recovery 89.4%∼106.5%) and sensitivity (LOD 0.02 âˆ¼ 0.98 ng/mL). Thus, the present response-boosting based protocol provides a promising application in quality control of food rich in gelatins.

Discovery of peptide biomarkers by label-free peptidomics for discrimination of horn gelatin and hide gelatin from Cervus nippon Temminck.

Gelatin and gelatin-based derivatives have been attracting worldwide attention as health-food ingredients. Deer horn gelatin (DCG), a well-known and expensive gelatin food in Asia, has suffered adulterants by adding deer-hide gelatin (DHG) in it. However, robust and effective methods which could differentiate DCG from DHG are still unavailable. This study is committed to discover peptide biomarkers to distinguish DCG from DHG using label-free peptidomics by nanoLC-MS/MS. Multivariate statistical analysis combined with glycosylation sites analysis of peptides was applied to visualize the difference between DCG and DHG. As a result, four peptide biomarkers for distinguishing DCG and DHG were confirmed and validated by UPLC-MS/MS and MRM mode, which was also used to calculate adulteration percentage in commercial samples. The presented strategy may be also particularly helpful in the in-depth authentication of food gelatins from different tissues of the same species.