JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
·
2017
·
IF 2.603
Wenhua Li,Bin Yang,Dongmei Zhou,Jun Xu,Wenjia Li,Wen-Chen Suen
Sodium dodecyl sulfate capillary electrophoresis (CE-SDS) has been widely used to separate and quantify monoclonal antibody (mAb) fragments and impurities. Two fragments with different masses smaller than monomer were observed in the CE-SDS analysis of a recombinant IgG1 mAb treated at 40 °C for 28 days. Reversed phase liquid chromatography mass spectrometry (RP–LC–MS) and trypsin digestion peptide mapping followed by LC–MS/MS analysis were employed to conclusively demonstrate that these antibody fragments were the degradation products derived from a Ser–Ser peptide bond cleavage at the heavy chain complementarity determining region (CDR). The total percentage of the cleaved antibody heavy chain was increased from 1.1% in the control sample to 4.6% in the forced degradation sample stored at 40 °C for 28 days by using CE-SDS analysis. Native size exclusion liquid chromatography (SEC-HPLC) was used to analyze the fragment profile of this treated mAb but did not reveal fragments smaller than the monomer. However, a small fragment was detected using denaturing size exclusion liquid chromatography (dSEC-HPLC) and was confirmed to be the N-terminal fragment of the heavy chain cleaved at the CDR region using RP–LC–MS analysis. These results suggested that this N-terminal fragment may be closely associated with the rest of the cleaved antibody molecule through non-covalent bond interactions. Moreover, when dSEC-HPLC and RP–LC–MS were used to analyze the aggregate and monomer samples individually collected from SEC-HPLC, both samples contained the same amount of the cleaved N-terminal fragment. These results suggested that the CDR region cleaved fragments likely not be involved in aggregates formation of this antibody. This case study reiterated that a combination of orthogonal analytical methods plays a crucial role in comprehensively analyzing and characterizing antibody fragmentation.
研究领域:
生物化学
·
分析化学
·
蛋白质组学
✓ 已验证
DOI
高影响因子
JOURNAL OF MASS SPECTROMETRY
·
2015
·Vol. 50
·期 1
·212-219
·
IF 2.379
Huixin Wang,Bing Wang,Zhonglin Wei,Hao Zhang,Xinhua Guo
A good understanding of gas-phase fragmentation chemistry of peptides is important for accurate protein identification. Additional product ions obtained by sodiated peptides can provide useful sequence information supplementary to protonated peptides and improve protein identification. In this work, we first demonstrate that the sodiated a 3 ions are abundant in the tandem mass spectra of sodium-cationized peptides although observations of a 3 ions have rarely been reported in protonated peptides. Quantum chemical calculations combined with tandem mass spectrometry are used to investigate this phenomenon by using a model tetrapeptide GGAG. Our results reveal that the most stable [a 3 + Na − H] + ion is present as a bidentate linear structure in which the sodium cation coordinates to the two backbone carbonyl oxygen atoms. Due to structural inflexibility, further fragmentation of the [a 3 + Na − H] + ion needs to overcome several relatively high energetic barriers to form [b 2 + Na − H] + ion with a diketopiperazine structure. As a result, low abundance of [b 2 + Na − H] + ion is detected at relatively high collision energy. In addition, our computational data also indicate that the common oxazolone pathway to generate [b 2 + Na − H] + from the [a 3 + Na − H] + ion is unlikely. The present work provides a mechanistic insight into how a sodium ion affects the fragmentation behaviors of peptides. Copyright © 2015 John Wiley & Sons, Ltd.
研究领域:
生物化学
·
质谱分析
·
理论化学
✓ 已验证
DOI
PubMed
高影响因子
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
·
2019
·
IF 2.813
Bin Yang,Wenhua Li,Hui Zhao,Anling Wang,Yuanjun Lei,Qiuling Xie,Sheng Xiong
Monoclonal antibody (mAb) fragmentation is a widespread issue of protein stability that needs to be carefully monitored for critical mAb quality control during the production process development. This study describes here the discovery and characterization of CHO host cell protease-induced fragmentation of a therapeutic mAb-X in the formulation samples from an early production process. The fragmentation was observed in the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) analysis of mAb-X formulation samples incubated at elevated temperature. Size exclusion liquid chromatography (SEC-HPLC) was used to analyze and collect these cleaved fragments derived from mAb-X. Reversed phase liquid chromatography mass spectrometry (RP-LC-MS) and tandem mass (MS/MS) analysis demonstrated that the fragment was generated mainly due to the hinge region cleavage of mAb-X. The fragmentation rate was characterized in the mAb-X formulation samples at pH from 4.0 to 6.0 using CE-SDS and SDS-PAGE analysis. The percentage of the main fragment increased dramatically from 2.8% to 31.2% as pH decreased from 6.0 to 4.0 at 40 °C for 28 days, which indicated the fragmentation was highly pH-dependent. The SDS-PAGE analysis further verified the pH-dependent property of the framentation of mAb-X. Moreover, the fragmentation was characterized in the presence and absence of pepstatin A, an inhibitor of acidic proteases. Significant inhibition of mAb-X fragmentation was observed with the addition of pepstatin A to mAb-X formulation samples. These results suggested residual acidic host cell protease(s) in the formulation samples from an early production process caused the fragmentation of mAb-X. To prove evidence, we developed an optimized protein A chromatography to enhance the residual host cell protease(s) removal capability of mAb-X purification process and consequently eliminate the above described cleaved fragment of mAb-X, which further supported the hypothesis that the fragmentation of mAb-X was catalyzed by the residual host cell protease(s) in the formulation samples from the early production process. This case study reiterated that residual host cell protease is a critical quality attribute (CQA) that should be carefully controlled and evaluated to guarantee successful manufacture processes for mAb products.
研究领域:
生物技术
·
蛋白质工程
·
制药科学
✓ 已验证
DOI
高影响因子
JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING
·
2024
·
IF 1.9
Yue Xiao,Shuai Yuan,Ruxin Luo,Yiling Tang,Xin Wang,Ping Xiang,Bin Di
The ketamine (KET) and its analogs consumed by humans are becoming emerging contaminants (ECs), as they at present in surface waters after being carried through wastewater systems. Drugs in wastewater can be analyzed using the direct-injection method, a simple wastewater analysis (WWA) method that can provide objective, continuous and nearly to real-time findings. This article describes an ultra-high-pressure liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the simultaneous quantification and confirmation of seven KET-based ECs in wastewater by direct injection. After optimization of the UPLC–MS/MS and sample pretreatment conditions, the method was validated and applied to samples (n = 157) collected from several wastewater treatment plants (WWTPs) in southern China in which KET had the highest detection rate. The established direct-injection method was not only simple to perform but also had better sensitivity, shorter detection times, and analyzed more KET-based ECs than currently published methods, meeting the requirements for the monitoring and high-throughput analysis of common KET-based ECs. We also analyzed the fragmentation pathway of KET-based ECs to obtain product ion information on other unknown substances. Additional studies are needed to establish a comprehensive direct-injection screening method of ECs in wastewater on model-based assessment.
研究领域:
环境科学
·
分析化学
·
毒理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Scientific Reports
·
2017
·Vol. 7
·期 1
·1-9
·
IF 4.259
Li Lanhui,Jin Mingliang,Sun Chenglong,Wang Xiaoxue,Xie Shuting,Zhou Guofu,van den Berg Albert,Eijkel Jan C. T.,Shui Lingling
DNA fragmentation down to a precise fragment size is important for biomedical applications, disease determination, gene therapy and shotgun sequencing. In this work, a cheap, easy to operate and high efficiency DNA fragmentation method is demonstrated based on hydrodynamic shearing in a bubbling system. We expect that hydrodynamic forces generated during the bubbling process shear the DNA molecules, extending and breaking them at the points where shearing forces are larger than the strength of the phosphate backbone. Factors of applied pressure, bubbling time and temperature have been investigated. Genomic DNA could be fragmented down to controllable 1–10 Kbp fragment lengths with a yield of 75.30–91.60%. We demonstrate that the ends of the genomic DNAs generated from hydrodynamic shearing can be ligated by T4 ligase and the fragmented DNAs can be used as templates for polymerase chain reaction. Therefore, in the bubbling system, DNAs could be hydrodynamically sheared to achieve smaller pieces in dsDNAs available for further processes. It could potentially serve as a DNA sample pretreatment technique in the future.
研究领域:
生物技术
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
TALANTA
·
2025
·
IF 6.1
Luke Wei,Yuxin Hu,Jiamin Wu,Jing Mao,Tianqin Yin,Jieqiong Qiu
Doxorubicin (DOX), a widely utilized chemotherapeutic antibiotic, increasingly threatens environmental and public health due to its persistence in marine ecosystems, bioaccumulation in organisms, and contamination of agricultural systems. In response to the urgent demand for effective DOX detection, we have developed a label-free fluorescent aptamer biosensor (FAB) that employs SYBR Green I (SG I)-mediated signal amplification. A key innovation in this design is the strategic fragmentation of the 41-mer complementary strand (CP) of the DOX aptamer into three 11-mer fragments. It significantly mitigates nonspecific SG I binding interference caused by the longer ssDNA CP, enhancing the fluorescence signal ratio (F dsDNA /F ssDNA ) from 4.6-fold to 12.6-fold. Upon DOX introduction, competitive binding between DOX, SG I, and DNA further suppresses background noise, yielding an exceptional fluorescence quenching ratio (F 0 /F) of 52.8. The optimized FAB demonstrates ultra-sensitive DOX detection, with a detection limit of 1.41 nM and a linear response between 1.41 nM and 300 nM. The recoveries of DOX spiked into pond water and drinking water samples ranged from 95.1% to 101.8%, with relative standard deviations (RSDs) below 3.46%, demonstrating negligible matrix effects and confirming the FAB method’s robustness for environmental monitoring applications. This strategy synergistically enhances signal transduction efficiency, and strengthens detection reliability, all while maintaining operational simplicity. As a result, this FAB for DOX detection holds transformative potential, poised to revolutionize biosensor technology in the fields of food safety, environmental monitoring, and biosecurity.
研究领域:
生物传感器
·
环境科学
·
分析化学
·
分子生物学
·
生物化学
·
纳米技术
·
公共卫生
·
海洋生态学
✓ 已验证
DOI
PubMed
高影响因子
Metallomics
·
2021
·Vol. 13
·期 10
·
IF 4.526
Ding Haoxuan,Zhang Qian,Yu Xiaonan,Chen Lingjun,Wang Zhonghang,Feng Jie
Iron overload is an important contributor to disease. The liver, the major site of iron storage in the body, is a key organ impacted by iron overload. While several studies have reported perturbations in liver lipids in iron overload, it is not clear, on a global scale, how individual liver lipid ions are altered. Here, we used lipidomics to study the changes in hepatic lipid ions in iron-overloaded mice. Iron overload was induced by daily intraperitoneal injections of 100 mg/kg body weight iron dextran for 1 week. Iron overload was verified by serum markers of iron status, liver iron quantitation, and Perls stain. Compared with the control group, the serum of iron-overload mice exhibited low levels of urea nitrogen and high-density lipoprotein (HDL), and high concentrations of total bile acid, low-density lipoprotein (LDL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), suggestive of liver injury. Moreover, iron overload disrupted liver morphology, induced reactive oxygen species (ROS) production, reduced superoxide dismutase (SOD) activity, caused lipid peroxidation, and led to DNA fragmentation. Iron overload altered the overall composition of lipid ions in the liver, with significant changes in over 100 unique lipid ions. Notably, iron overload selectively increased the overall abundance of glycerolipids and changed the composition of glycerophospholipids and sphingolipids. This study, one of the first to report iron-overload induced lipid alterations on a global lipidomics scale, provides early insight into lipid ions that may be involved in iron overload-induced pathology.
研究领域:
肝病学
·
脂质组学
·
病理学
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
NEW JOURNAL OF CHEMISTRY
·
2024
·
IF 2.7
Shuwei Shen,Binbin Huang,Zhiyi Peng,Luting Yu,Lili Ren,Jia Liu,Guoguang Chen
Traditional Chinese medicine (TCM), as a significant complementary and alternative approach, has received increasing attention from medical practitioners worldwide and is now regarded as an important source for modern drug discovery owing to its special therapeutic efficiency and few side effects. In this work, a popular TCM, Ganoderma lingzhi (G. lingzhi), was used as a test drug to show how Raman spectroscopy combined with traditional chemical and biological assays could be used to monitor alternations in molecular events in G. lingzhi extract-induced cells with high sensitivity. The observations indicated that the Raman fingerprint of MDA-MB-231 cells after G. lingzhi triterpene treatment was significantly different in contrast to that of the control, and changes in the Raman bands showed that some important molecular events could be discerned, including protein denaturation, DNA fragmentation, and lipid metabolism disorder. Network pharmacology analysis further supplemented the drug's target and signaling pathways. Our work proposes an effective way for screening potential new drug products early from the complex chemical compositions of TCM, as well as for accelerating preclinical medicinal chemistry optimization cycles, playing a significant role in the development of new drugs.
研究领域:
药理学
·
光谱学
·
细胞生物学
·
中药
·
药物发现
✓ 已验证
产品关联
DOI
高影响因子
Molecular Medicine Reports
·
2015
·Vol. 12
·期 3
·3869-3873
·
IF 1.554
Shi‑Min Yu,Dong‑Hui Hu,Jian‑Jun Zhang
Hepatocellular carcinoma (HCC) is a highly malignant tumor, associated with poor patient prognoses, and high rates of morbidity and mortality. To date, the therapeutic strategies available for the treatment of HCC remain limited. The present study aimed to elucidate the anticancer activity of umbelliferone, a naturally occurring coumarin derivative isolated from Ferula communis, against the HepG2 HCC cell line. A 3‑(4,5‑dimthylthaizol‑2‑yl)‑2,5, diphenyltetrazolium bromide assay was used to evaluate cell viability following umbelliferone treatment, and the effects of umbelliferone on cell cycle progression and apoptosis were evaluated using flow cytometry. The presence of morphological features characteristic of apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation, were evaluated in HepG2 cells following umbelliferone treatment. Cell cycle analysis conducted via propidium iodide (PI) staining indicated that umbelliferone treatment induced cell cycle arrest at S phase in HepG2 cells. Analysis with Annexin V and PI staining revealed that umbelliferone induced apoptotic events in HepG2 cells in a concentration‑dependant manner (0‑50 µM). Umbelliferone also induced dose‑dependant DNA fragmentation. In conclusion, umbelliferone was found to exhibit significant anticancer effects via the induction of apoptosis, cell cycle arrest and DNA fragmentation in HepG2 cancer cells.
研究领域:
肿瘤学
·
药理学
·
细胞生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Food Bioscience
·
2019
·
IF 3.22
Chun-Min Ma,Tie-Jing Li,Xin-Huai Zhao
Lactoferrin hydrolysates (LF-H) were digested using pepsin and modified using a pepsin-catalyzed plastein reaction with tryptophan (Trp) to obtain a target product. LF-H, the target product, plastein reaction-modified LF-H alone, and a mixture of LF-H and Trp were evaluated for their activities with human gastric cancer BGC-823 cells at 24 and 48 h. Assay results indicated that the target product had decreased free amino groups and had incorporated Trp. LF-H and the plastein reaction-modified LF-H alone did not show detectable growth inhibition on BGC-823 cells at 1–3 mg/mL; however, the target product at the same dose levels inhibited cell growth, and showed higher activity than the mixture ( P < 0.05). The target product at 2 and 3 mg/mL stopped cell cycle progression at the G0/G1-phase, induced DNA fragmentation , and showed apoptosis induction, which increased the number of apoptotic cells. Moreover, time- and dose-dependent responses were observed in these experiments. RT-PCR assay results indicated that exposure of the cells to the target product resulted in up- and down-regulated mRNA expression for 7 apoptosis-related genes, namely Bcl-2, Bcl-xl, Bad, Bax, caspase-3, caspase-6, and caspase-9. Thus, the plastein reaction increased in vitr o anticancer activities of LF-H with BGC-823 cells, and more importantly, both the plastein reaction and Trp incorporation contributed to enhanced activities.
研究领域:
生物化学
·
癌症研究
·
分子生物学
✓ 已验证
产品关联
DOI
高影响因子
ANIMAL REPRODUCTION SCIENCE
·
2025
·
IF 3.3
Chaoyue Cao,Xue Qin,Yiwen Sha,Haoqi Xiao,Zhenheng Lai,Yi Zheng,Rui Cai,Weijun Pang
Vitamin E (VE) is widely used as an antioxidant in boar semen preservation. Its low water solubility and limited bioavailability, however, compromise its functional efficacy during storage. To address these limitations, we developed a lecithin-stabilized vitamin E nanoemulsion (NE) to enhance its dispersibility and antioxidant efficacy. Semen from Duroc boars was diluted in Beltsville thawing solution (BTS) containing either 0.6 mM NE, 4 mM free VE, or no additive, and stored at 17 °C. Compared to VE and control groups, NE significantly enhanced sperm motility, preserved the structural integrity of the plasma and acrosomal membranes, and maintained mitochondrial function, while reducing oxidative stress and DNA fragmentation over a 7d storage period ( P < 0.01). Untargeted metabolomics revealed that NE supplementation regulated key pathways including unsaturated fatty acid metabolism and glutathione-related antioxidation. Artificial insemination (AI) trials involving 637 sows confirmed that NE-treated semen improved total and healthy piglet numbers per litter ( P < 0.05), without adverse effects. These findings suggest that NE provides an effective and practical strategy for enhancing boar semen preservation and reproductive outcomes in porcine production.
研究领域:
动物繁殖
·
纳米技术
·
抗氧化研究
·
代谢组学
·
兽医学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
WATER RESEARCH
·
2020
·
IF 9.13
Hangbo Xu,Yupan Zhu,Mengru Du,Yuqi Wang,Siyao Ju,Ruonan Ma,Zhen Jiao
Although the identification of effective reactive oxygen species (ROS) generated by plasma has been extensively studied, yet the subcellular mechanism of microbial inactivation has never been clearly elucidated in plasma disinfection processes. In this study, subcellular mechanism of yeast cell inactivation during plasma-liquid interaction was revealed in terms of comprehensive factors including cell morphology, membrane permeability, lipid peroxidation, membrane potential, intracellular redox homeostasis (intracellular ROS and H 2 O 2 , and antioxidant system (SOD, CAT and GSH)), intracellular ionic equilibrium (intracellular H + and K + ) and energy metabolism (mitochondrial membrane potential, intracellular Ca 2+ and ATP level). The ROS analysis show that ·OH, 1 O 2 , ·O 2 − and H 2 O 2 were generated in this plasma-liquid interaction system and ·O 2 − served as the precursor of 1 O 2 . Additionally, the solution pH was reduced. Plasma can effectively inactivate yeast cells mainly via apoptosis by damaging cell membrane, intracellular redox and ion homeostasis and energy metabolism as well as causing DNA fragmentation. ROS scavengers ( l -His, d -Man and SOD) and pH buffer (phosphate buffer solution, PBS) were employed to investigate the role of five antimicrobial factors (·OH, 1 O 2 , ·O 2 − , H 2 O 2 and low pH) in plasma sterilization. Results show that they have different influences on the aforementioned cell physiological activities. The ·OH and 1 O 2 contributed most to the yeast inactivation. The ·OH mainly attacked cell membrane and increased cell membrane permeability. The disturb of cell energy metabolism was mainly attributed to 1 O 2 . The damage of cell membrane as well as extracellular low pH could break the intracellular ionic equilibrium and further reduce cell membrane potential. The remarkable increase of intracellular H 2 O 2 was mainly due to the influx of extracellular H 2 O 2 via destroyed cell membrane, which played a little role in yeast inactivation during 10-min plasma treatment. These findings provide comprehensive insights into the antimicrobial mechanism of plasma, which can promote the development of plasma as an alternative water disinfection strategy.
研究领域:
微生物学
·
等离子体物理
·
环境科学
·
细胞生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）