List of Issues

Chinese Journal of Chromatography
2021, Vol. 39, No. 10
Online: 08 October 2021

Previous Issue    Next Issue



For Selected:

Download Citations EndNote Ris BibTeX

Toggle Thumbnails

Editorial

Select

Editorial for Special Issue for the Celebration of the 80th Birthday of Academician ZHANG Yukui (Ⅱ) Editorial Office of Chinese Journal of Chromatography 2021, 39 (10):  1039-1040.  DOI: 10.3724/SP.J.1123.2021.08025 Abstract ( 52 )   HTML ( 296 )   PDF (490KB) ( 62 )

Highlights

Select

Advances in materials for circulating tumor cells capture SUN Wenjing, SHI Zhenqiang, QING Guangyan 2021, 39 (10):  1041-1044.  DOI: 10.3724/SP.J.1123.2021.05020 Abstract ( 147 )   HTML ( 208 )   PDF (2344KB) ( 107 )

Reviews

Select

Recent advances in glycopeptide enrichment and mass spectrometry data interpretation approaches for glycoproteomics analyses LIU Luyao, QIN Hongqiang, YE Mingliang 2021, 39 (10):  1045-1054.  DOI: 10.3724/SP.J.1123.2021.06011 Abstract ( 388 )   HTML ( 222 )   PDF (2031KB) ( 220 )   Protein glycosylation is one of the most important post-translational modifications (PTMs). The glycosylation is crucial in a variety of physiological and pathological processes that include protein stability, intracellular and intercellular signal transduction, hormone activation or inactivation, and immune regulation. Protein glycosylation is generated by complex biosynthetic pathways comprising hundreds of glycosyltransferases, glycosidases, transcriptional factors, transporters, and protein backbones. Abnormal protein glycosylation is closely associated with the occurrence and development of diseases. Many disease biomarkers in clinical screening are glycoproteins (alfa fetoprotein for liver cancer, carbohydrate antigen 125 for ovarian cancer, carcinoembryonic antigen for colon cancer, prostate-specific antigen for prostate cancer, etc.), and glycan antigens (carbohydrate antigen 19-9 for gastrointestinal cancer and pancreatic cancer, etc.). Glycoproteomics research and technological developments are important to elucidate the mechanism of protein glycosylation in vivo. Mass spectrometry (MS)-based proteomics provides an excellent approach for the comprehensive analysis of proteins and their modifications. In bottom-up proteomics, glycoproteomic analysis is more difficult than other PTMs because intact glycopeptides have diverse peptide backbones and glycan chains, relatively low abundance and ionization efficiency, and pronounced heterogeneity. In recent years, glycoproteomic methodologies such as intact glycopeptide enrichment methods, MS fragmentation and acquisition approaches, MS data interpretation tools and software, and quantification strategies have been appreciably improved. These methodologies have driven in-depth glycoproteomics research. This review focuses on the recent advances in MS-based glycoproteomics. New enrichment methods and spectral interpretation approaches of intact N- and O-glycopeptides are discussed. Their applications in answering various questions in complex biological systems are also considered. The new enrichment methods for intact glycopeptides are mostly based on existing principles. Some properties of the materials, such as hydrophilicity and electrophilicity, have been optimized to improve the enrichment performance. For example, dual-functional Ti(IV)-IMAC materials have been used for the separation of glycopeptides and phosphopeptides. Considering the clinical applications, some glycoproteomics methods integrate enrichment processing into automated workflows to reduce errors caused by manual operations and to increase the experimental reproducibility and efficiency. For example, an automated glycopeptide enrichment method consisting of a liquid chromatograph equipped with a hydrophilic interaction chromatography column has been shown capable of highly reproducible analyses of site-specific glycopeptides in complex biological samples. These methods are more suitable for the discovery of newly glycosylation-related biomarkers as well as for the physiopathological studies of human diseases. With the optimization of glycopeptide enrichment methods and the innovation of MS technologies in the past decade, MS analysis of intact glycopeptides has begun to yield a wealth of glycopeptide fragment ions and plentiful high-quality MS data. This review introduces several effective fragmentation methods for intact glycopeptides. These include collision-induced dissociation, high-energy collision dissociation, electron capture dissociation, electron-transfer dissociation, and electron-transfer/higher-energy collision dissociation. Automated analysis of MS data of intact N- and O-glycopeptides requires interpretation approaches and corresponding software tools with high sensitivity and reliability. Finally, we highlight the utility of several spectral interpretation approaches and their corresponding popular search software, including ArMone, Byonic, GPQuest, pGlyco, O-search, MSFragger-Glyco, and O-Pair Search. In addition, MS data acquisition modes, such as data-dependent acquisition, data-independent acquisition, multiple reaction monitoring technology, and parallel reaction monitoring technology, have great application prospects in glycoproteomics research. With the improvements in enrichment methods, MS technologies, and spectral interpretation approaches for intact N- and O-glycopeptides, comprehensive and systematic glycoproteomics analysis has tremendously expanded the knowledge of protein glycosylation. These glycoproteomic technologies have a wide range of applications that include exploring the molecular mechanisms of protein glycosylation and discovering the new biomarkers of human diseases.

Select

Comparison and progress review of various super-resolution fluorescence imaging techniques CHEN Jie, LIU Wenjuan, XU Zhaochao 2021, 39 (10):  1055-1064.  DOI: 10.3724/SP.J.1123.2021.06015 Abstract ( 952 )   HTML ( 212 )   PDF (3524KB) ( 380 )   “Seeing is believing” is the central philosophy of life science research, which runs through the continuous understanding of individual molecules, molecular complexes, molecular dynamic behavior, and the entire molecular network. Living and dynamic molecules are functional in nature; therefore, fluorescence microscopy has emerged as an irreplaceable tool in life science research. However, when fluorescence imaging is performed at the molecular level, some artificial signals may lead to erroneous experimental results. This obstacle is due to the limitation of the optical diffraction limit, and the fluorescence microscope cannot distinguish the target in the diffraction-limited space. Super-resolution fluorescence imaging technology breaks through the diffraction limit, allows visualization of biomolecules at the nanometer scale to the single-molecule level, and allows us to study the structure and dynamic processes of living cells with unprecedented spatial and temporal resolution. It has become a powerful tool for life science research and is gradually being applied to material science, catalytic reaction processes, and photolithography as well. The principle of super-resolution imaging technologies is different; therefore, it has different technical performances, thus limiting their specific technical characteristics and application scope. Current mainstream super-resolution imaging technologies can be classified into three types: structured illumination microscopy (SIM), stimulated emission depletion (STED), and single-molecule localization microscopy (SMLM). These microscopes use different complex technologies, but the strategy is the same and simple, i.e. two adjacent luminous points in a diffraction-limited space can be spatially resolved by time resolution. SIM has been used for three-dimensional real-time imaging in multicellular organisms; however, compared with other technologies, its lower horizontal and vertical resolutions need to be further optimized. STED is limited by its small imaging field of view and high photobleaching; however, the best time resolution can be considered at a high spatial resolution, and it has been proven that three-color STED imaging can be performed. In SMLM super-resolution imaging, the time resolution is affected by the time required to locate all fluorophores, which is closely related to the switching and luminescence properties of the fluorophore. With the improvement in horizontal and vertical resolution of imaging, the image acquisition speed, photobleaching characteristics, and the possibility of multi-color and dynamic imaging have increasingly become the key determinants of super-resolution fluorescence imaging. Thus far, the main use of super-resolution imaging technology has been focused on biological applications for studying structural changes less than 200 nm in dimension. In addition to the combination of structural and morphological characterization with biomolecular detection and identification, super-resolution imaging technology is rapidly expanding into the fields of interaction mapping, multi-target detection, and real-time imaging. In the latter applications, super-resolution imaging technology is particularly advantageous because of more flexible sample staining, higher labeling efficiency, faster and simpler readings, and gentler sample preparation procedures. In this article, we compare the principles of these three technologies and introduce their application progress in biology. We expect the results described herein will help researchers clarify the technical advantages and applicable application directions of different super-resolution imaging technologies, thus facilitating researchers in making reasonable choices in future research.

Select

Research advances in nano liquid chromatography instrumentation YANG Sandong, LI Naijie, MA Zhou, TANG Tao, LI Tong 2021, 39 (10):  1065-1076.  DOI: 10.3724/SP.J.1123.2021.06017 Abstract ( 519 )   HTML ( 202 )   PDF (2764KB) ( 242 )   The miniaturization of liquid chromatography equipment is among the most important focus areas in chromatographic technology. It involves the miniaturization of the physical dimensions of the instrument, size of the separation material, and inner diameter of the column. The advantages of a reduced inner diameter of the column have been investigated for several decades, and can be summarized as follows. First, the sample consumption is lower, which is particularly beneficial when a limited amount of sample is available, as is the case with natural products, and in biochemistry and biomedicine. Second, the consumption of the mobile phase is reduced, making the process environmentally friendly and facilitating green chemistry. This allows the addition of more expensive solvent additives, such as chiral additives or isotopic reagents, while maintaining a low analysis cost. Moreover, the degree of band dilution in the column is lower than that with conventional liquid chromatography under the same sample injection conditions. Thus, enhanced mass sensitivity is achieved. Other benefits of a reduced inner diameter of the column include temperature control due to effective heat transfer through the columns and easier coupling to mass detectors, which is particularly advantageous for analyzing complex samples. Typically, the term “nano liquid chromatography” is associated with liquid chromatography, which employs capillary columns of inner diameters less than 100 μm and flow rates in the range of tens to hundreds of nanoliters per minute. Because of the extremely low flow rates and small column volume, the extra-column effect becomes more prominent. Thus, the requirements for every component of liquid chromatographs are augmented toward improving their performance and optimizing the extra-column band broadening of the entire system. The solvent delivery equipment should be able to pump mobile phases accurately and steadily at nanoliter-level flow rates. A gradient mode is required to achieve this, which implies that the lowest flow rate for a single pump unit should reach a few nanoliters per minute. A certain operating pressure is also necessary to employ columns with different inner diameters and particle sizes. A precise and repeatable sample injection procedure is essential for nano liquid chromatography. The injection volume and mode should be suitable for capillary columns, without inducing a significant extra-column effect. A higher-sensitivity detector should be employed, and sample dispersion should be limited. The improved tubing and connection method in nano liquid chromatography should offer stability, reliability, and ease of operation. The extra-column volume should also be restricted to suit nanoliter-level flow rates. Considering that most nano liquid chromatographic instruments have been coupled with a mass detector, this review mainly focused on nanoliter solvent delivery modules, sample injection modules, and tubing and connection modules. By searching and summarizing research articles, technical patents, and brochures of instrument manufacturers, technical routes and research progress on these modules were described in detail. The pump designs can be classified into four types. Pneumatic amplifying pumps have been used in ultra-high-pressure applications. The flow-splitting delivery system, though easy to realize, may lead to a large amount of solvent wastage. Splitless pumps, which are classified based on two main principles, are widely used. Some pumps based on other physical phenomena have been suggested; however, they lacked stability and robustness. Two types of injection modes have been utilized in nano liquid chromatography. The direct nanoliter injection mode typically takes advantage of the groove on the rotor of a switching valve. The trapping injection mode uses trap columns to enable the introduction of large sample volumes. As for the tubing and connection, a few appropriate designs can be acquired from commercial suppliers. The robustness has been improved using some patented technologies. The optimization principles and research progress on optical absorption detection are briefly introduced. Finally, commercial nano liquid chromatographic systems are compared by considering the pumps and injectors.

Select

Advances in chromatography in the study of drug-plasma protein interactions BAI Yu, FAN Yufan, GE Guangbo, WANG Fangjun 2021, 39 (10):  1077-1085.  DOI: 10.3724/SP.J.1123.2021.06028 Abstract ( 259 )   HTML ( 200 )   PDF (1512KB) ( 115 )   After entering human blood circulation, small-molecule drugs interact extensively with various plasma proteins, such as human serum albumin and α1-acid glycoprotein. These interactions profoundly affect the distribution of drugs in vivo and the binding of drugs to targets, thus affecting the efficacy of drugs. In-depth investigation of drug-plasma protein interactions is of great significance for the optimization of drug properties, the development of new drugs, risk assessment, and combination therapy of drugs. Therefore, it is essential to develop highly efficient, sensitive, and accurate methods for elucidating drug-plasma protein interactions. Chromatography is a powerful tool with high throughput, high separation performance, and high sensitivity in the characterization of drug-protein interactions. High-performance affinity chromatography (HPAC) and capillary electrophoresis (CE) have been widely utilized in this field. These methods include the determination of the effects of the posttranslational modification of proteins on binding and the competitive binding of multiple drugs. In addition, various chromatographic methods are used to obtain interaction information such as the binding constant, binding-site number, and dissociation rate constant. In this review, the common strategies and recent advances in HPAC and CE in the study of drug-plasma protein interactions are briefly reviewed. The immobilization methods of proteins, the principles and applications of frontal analysis, zonal elution, ultrafast affinity extraction, peak profiling, and peak decay analysis are discussed for HPAC and affinity capillary electrophoresis (ACE) and capillary electrophoresis frontal analysis (CE-FA) for CE. HPAC relies on the fixation of proteins on the surfaces of chromatographic stationary phases by covalent linking or physical adsorption, followed by obtaining the drug-protein interaction information through a variety of chromatographic methods. In the frontal chromatography analysis, mobile phases with different concentrations of drugs are passed through the HPAC column to obtain different breakthrough times. The process can determine the number of drug binding sites and the binding constant of each site in the affinity protein with high accuracy. The zonal elution method can detect the drug binding sites on proteins using site-specific probes to determine whether there is competition between drugs and probes. The sample consumption and analysis time of the zonal elution method are much less than those in frontal chromatography analysis. The ultrafast affinity extraction method can inject complex samples, such as serum, into affinity columns to determine the free drug components. It can measure the combination and dissociation constants of drug-protein interactions by changing the chromatography flow rate. Peak profiling and peak decay analyses are both effective methods for investigating the dissociation of drugs and proteins. In CE analysis, the drug and protein samples are dissolved in an electrophoresis buffer, and their interactions are measured during electrophoresis with high accuracy and low sample consumption. However, the adsorption of proteins on the capillary wall can compromise CE performance. Common CE methods in drug-protein interaction analysis are ACE and CE-FA. ACE is usually performed by changing the effective mobility of drugs via the addition of different concentrations of proteins. This method has been widely used, and several variant techniques have been developed recently. CE-FA involves the sampling of a drug premixed at a known concentration with a target protein. Compared with other CE methods, CE-FA exhibits the unique advantages of high throughput, automatic online analysis, and the ability to determine high-order drug-protein interactions. Finally, the shortcomings of current chromatography methods are summarized, and the application prospects and development direction of chromatography technology in the field of drug-plasma protein interaction research are discussed.

Articles

Select

Comparison of the performance of secretome analysis based on metabolic labeling by three unnatural sugars MAO Yuan, ZHENG Jiangnan, FENG Shun, TIAN Ruijun 2021, 39 (10):  1086-1093.  DOI: 10.3724/SP.J.1123.2021.04017 Abstract ( 194 )   HTML ( 195 )   PDF (3061KB) ( 102 )   Many secreted proteins, including cytokines, growth factors and hormones, are crucial in processes like intercellular signaling. Dynamic changes in secreted proteins usually reflect the growth and pathological state of the cells. Many drug targets are secretory proteins. The proteins are also important biomarkers. Conditioned cell culture media are important samples for secretory proteomic studies. Biomass spectrometry-based proteomic analysis enables the systematic study of secretory proteins. The main problem in analyzing secretory proteins in conditioned culture media is the low concentration of these proteins and the presence of serum, amino acids, and additives in culture media that may interfere with the protein analysis. Conventional secretory proteome analysis uses serum-free cell culture to reduce sample complexity, and typically involves protein concentration, purification, and desalting using ultrafiltration, dialysis, lyophilization, and trichloroacetic acid (TCA) or acetone precipitation, followed by enzymatic digestion and mass spectrometry analysis. This analytical process does not allow specific enrichment of secreted proteins. Thus, only a few secreted proteins can be identified. In addition, prolonged serum-free incubation of cells also tends to lead to unexpected changes in their activity status. A bioorthogonal-based enrichment approach can effectively avoid this problem. In recent years, unnatural sugars containing bio-orthologous groups, such as azide groups, have been used to metabolically label glycosylated proteins, enabling cellular imaging or selective enrichment of glycoproteins and their use for proteomic analysis. The strategy is a two-step process. First, azide-based sugar analogues are added to the cell culture medium and introduced to glycoproteins via the intracellular glycan biosynthesis pathway. Second, they are specifically covalently labeled with imaging probes or affinity probes via click chemistry. Since secreted proteins are usually glycoproteins, this glycolytic labeling has been used to label and enrich secreted proteins. N-Azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz), and N-azidoacetylmannosamine (ManNAz) are classical azide-based sugar analogues. Their effects on cytoplasmic membrane proteins have been compared. However, only ManNAz has been used for metabolic labeling of secreted proteins. No other glyco-analogues that label secreted proteins have been reported. Here, the bio-orthogonal chemical biology technology achieved highly selective labeling and enriched secreted proteins. In combination with click chemistry, different sugar analogues were evaluated for metabolic labeling of secreted proteins. HeLa cells were metabolically labeled by ManNAz, GalNAz, and GlcNAz (the three most commonly used commercial sugar analogues). These glycolytic markers can selectively label specific types of glycosylation. For example, ManNAz, an analogue of the biosynthetic precursor of sialic acid, N-acetylmannosamine (ManNAc), can label sialylated N- or O-glycoproteins. GalNAz, an analogue of N-acetylgalactosamine (GalNAc), can replace GalNAc as a core residue of mucin-type O-glycans and thus label O-glycoproteins. In addition, the intracellular metabolic intermediate of GalNAz (pyrophosphate) UDP-GalNAz can be interconverted with UDP-GlcNAz catalyzed by UDP-galactose-4-differential isomerase (GALE) and thus can also label N-glycoproteins and O-GlcNAc glycoproteins instead of GlcNAc. The GlcNAz analogue is commonly used to label nuclear and cytoplasmic glycoproteins with β-O-GlcNAc residues, but can also label N-glycoproteins with mucin-type O-glycoproteins by converting GALE to GalNAz, followed by enrichment using a biotin-alkynyl probe. Label-free quantitative proteomic analysis was performed to evaluate their labeling efficiency. ManNAz-based secretory protein labeling identified 282 secretory proteins, 224 plasma membrane proteins, and 846 N-glycosites. Compared with GalNAz and GlcNAz, the enrichment of secreted proteins was increased 130% and 67.2%, respectively, and the enrichment of plasma membrane proteins was increased 273.3% and 148.7%, respectively. This study provides a useful comparative analysis and new strategies for highly selective enrichment and systematic secretome analysis.

Select

Extraction and isolation of histones from paraffin-embedded tissues and quantitative analysis of post-translational modifications TIAN Shanshan, LIU Ranran, QIAN Xiaolong, GUO Xiaojing, ZHANG Kai 2021, 39 (10):  1094-1101.  DOI: 10.3724/SP.J.1123.2021.06018 Abstract ( 183 )   HTML ( 195 )   PDF (3407KB) ( 126 )   Histone post-translational modifications (HPTMs) have been believed to play crucial roles in the regulation of gene transcription. Thus, aberrant modification of histone can contribute to the occurrence and development of diseases such as tumors. To date, formalin fixed paraffin-embedded (FFPE) clinical tissues are believed to be one of the most valuable sample resources in the study of human diseases. Therefore, it is of great significance to reveal the molecular mechanism of cancer and discover the markers of tumor. Proteomics, based on high performance liquid chromatograph-tandem mass spectrometry (HPLC-MS/MS), has become a powerful tool for HPTM identification. However, HPTM analysis of FFPE samples is yet to be explored; it has not been reported in China to our best knowledge. In this study, a new method based on HPLC-MS/MS was developed for the extraction and separation of histone proteins and analysis and quantification of HPTMs in FFPE tissues. First, the strategy for the extraction and separation of histone proteins from FFPE samples were optimized. After comparing the extraction efficiency of two different methods, which include the acid extraction of histone and extraction of total protein, a novel method was developed for histone extraction, separation, and HPTMs analysis by integrating dewaxed hydration treatment of FFPE tissues with protein extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. Furthermore, the effects of operation parameters on histone extraction and HPTM identification were investigated, including number of paraffin embedded sections and chemical derivation of histone proteins. Thereafter, the identification and quantification of HPTMs were performed using reversed-phase HPLC-MS/MS in data independent acquisition (DIA) mode. Finally, the optimized methods were applied to quantitative analysis of HPTMs in FFPE tissues. A variety of HPTMs were identified; they included lysine methylation, acetylation, crotonylation, etc. Therefore, the spectrum of HPTMs on global level was set for human breast cancer and paracancerous tissues from two cases of FFPE clinical tissues. The sites holding differential HPTM level in cancer and paracancerous tissues were further obtained by quantifying the individual HPTMs. Thus, the relationship between HPTM level and tumor was discussed. Abnormal HPTM sites were characterized using cluster analysis, thus their similar tendency was found. For example, abnormal HPTM sites such as H3K9me3, H3K9ac, and H3K27me3 in cancers are associated with epigenetic regulation. It indicated that different epigenetic events might occur in cancer and paracancerous tissues. Interestingly, we found that the level of H4K20me3 were both significantly down-regulated in the two cancer tissues. HPTM had been thought to be a potential biomarker in breast cancer; therefore, these positive results indicated that our method is effective for HPTMs of clinical FFPE samples. Our study provides a useful tool for the isolation and analysis of HPTMs in clinical FFPE samples, showing the potential for the detection of epigenetic biomarker in cancer.

Select

Preparation of multi-functional magnetic nanoparticles for harvesting low-molecular-weight glycoproteins DOU Peng, XIANG Yumiao, LIANG Liang, LIU Zhen 2021, 39 (10):  1102-1110.  DOI: 10.3724/SP.J.1123.2021.07019 Abstract ( 142 )   HTML ( 185 )   PDF (2362KB) ( 87 )   Supporting Information Low-molecular-weight glycoproteins (LMW-GPs) are considered promising candidates for disease biomarker discovery. Selective sorbents are essential for the extraction and enrichment of this class of compounds. Boronate affinity chromatography is a unique separation mode in liquid chromatography. It enables the selective separation and isolation of cis-diol-containing compounds such as glycoproteins and saccharides. Recent years have witnessed the rapid development of boronate affinity materials, particularly for use as selective sorbents in proteomics and metabolomics. However, studies are scarce on the specific design of such materials for the selective extraction of LMW-GPs. Herein, we present multifunctional magnetic nanoparticles (MNPs) for selectively harvesting LWM-GPs. The multifunctional MNPs were rationally designed and prepared by wrapping magnetic core nanoparticles with a phenylboronic acid-grafted poly(acrylic acid) (PAA) network. In addition to fulfilling the primary function of conventional MNPs in magnetic separation, multifunctional MNPs can offer three pre-determined advanced functions: 1) the size-restriction effect, which enables the elimination of the interference of high-molecular-weight proteins and other species; 2) the selective extraction of LMW-GPs; and 3) protection of the harvested LMW-GPs against degradation and contamination. The multifunctional MNPs enable selective extraction due to the affinity of the boronic acid ligand to the cis-diol moieties of the glycoproteins. The size-restriction effect and protection function depend on the polymer network on the surface of the MNPs, which allows the selective passage of low-molecular-weight molecules. Transmission electron microscopy (TEM) characterization showed that the MNPs were well-shaped nanoparticles, with a diameter of approximately 60 nm. The size-restriction effect was first predicted by a thermogravimetric analysis-based theoretical calculation, where for MNPs prepared using PAA with an average molecular weight of 240 kDa, the estimated pore size of the network was 0.9 nm. The boronate affinity and size-exclusion effect were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillary zone electrophoresis (CZE). To investigate the dependence of the selectivity of the MNPs to LMW-GPs in a complex environment and the size-restriction threshold for the PAA chain length, nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) was performed to analyze the molecular mass of fragments harvested by the MNPs from the tryptic digest of horseradish peroxidase (HRP, a typical glycoprotein). The polymer chain length or the molecular weight of the PAA used played a critical role in determining the molecular weight thresholds of proteins above which the size exclusion effect will occur. The threshold values were found to be 5.0, 9.3, 4.1, 5.1, and 2.7 kDa for MNPs prepared using PAA with average molecular weights of 2, 5, 15, 100, and 240 kDa, respectively. This dependence enabled adjustment of the threshold value for inducing the size-exclusion effect of the multifunctional MNPs by changing the PAA chain length. The multifunctional MNPs can be further developed into promising nanoprobes for selectively harvesting not only LMW-GPs, but also other cis-diol-containing biomolecules of biological importance, such as nucleosides and glycans. Thus, the material preparation strategy reported herein offers new insights for the rational design and synthesis of multifunctional-affinity sorbents to selectively extract target compounds from a complex sample matrix.

Select

Preparation of Fe3O4@BA-MOF magnetic solid-phase extraction material and its application to the detection of pesticide residues in tea WANG Fengya, FENG Liang 2021, 39 (10):  1111-1117.  DOI: 10.3724/SP.J.1123.2021.06003 Abstract ( 163 )   HTML ( 195 )   PDF (3917KB) ( 115 )   Tea is one of the most popular beverages worldwide, and its quality is often affected by the excessive pesticide residues during production. During the detection of pesticide residues in tea by chromatography-mass spectrometry and other methods, a strong matrix effect attributed to tea polyphenols and pigments is observed, which seriously impacts the analysis results. In this study, Fe3O4 magnetic nanoparticles and boric acid-functionalized metal organic framework (BA-MOF) materials were combined to prepare a highly efficient adsorbent Fe3O4@BA-MOF for capturing tea polyphenols and pigments. An effective analysis method for pesticide residues in tea samples in combination with gas chromatography-mass spectrometry was established. The introduction of boronic acid ligands into the metal organic framework, as the recognition site of cis-diols, enhanced the polyphenol capture ability. Adsorption of the pigment in the matrix was achieved through π-π interactions between the MOF ligand and the pigment. This new material has significant advantages such as rapid magnetic separation, large surface area, and abundant functional sites. Fe3O4@BA-MOF was prepared by employing simple conditions and characterized by Fouriertransform infrared spectroscopy, scanning electron microscopy, and X-ray diffractometry to identify its functional groups and morphology. After investigating the adsorption effect of different doses of Fe3O4@BA-MOF adsorbents (5, 10, 30, 50, and 80 mg) on tea polyphenols, 50 mg of the adsorbent was added to the tea matrix and shaken thoroughly. The tea polyphenol content in the matrix solution was determined using an ultraviolet spectrophotometer. The polyphenols were reduced by 74.58% within 5 min. The effect of solution pH (2.0, 4.0, 6.0, and 7.0) on the adsorption efficiency was investigated, and pH 7.0 was chosen as the optimal condition. By adjusting the pH of the solution, Fe3O4@BA-MOF could be recycled, and it maintained the excellent adsorption performance after four cycles of use. The introduction of Fe3O4 magnetic nanoparticles led to rapid magnetic response characteristics during sample pretreatment and improved the pretreatment efficiency. In the actual application of tea pesticide detection, after Fe3O4@BA-MOF pretreatment, the average recovery rates of the ten pesticides were in the range of 75.8%-138.6%, and the RSD was in the range of 0.5%-18.7% (n=3). The Fe3O4@BA-MOF nanocomposite prepared by introducing the boric acid ligand into the MOF structure and incorporating Fe3O4 magnetic nanoparticles could specifically adsorb the tea polyphenol matrix. When applied to the detection of pesticide residues in tea, it purifies the matrix and improves the detection efficiency, thus being suitable for the detection and analysis of pesticides in tea.

Select

Identification of LAMTOR1-regulated metabolites using ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry in malignant transformation of liver inflammation WANG Wen, CHEN Di, PIAO Hailong 2021, 39 (10):  1118-1127.  DOI: 10.3724/SP.J.1123.2021.06006 Abstract ( 135 )   HTML ( 194 )   PDF (5121KB) ( 72 )   The late endosomal/lysosomal adaptor MAPK and mTOR activator 1 (LAMTOR1) is an important regulator protein in the response to energy stress. Public gene expression data shows that the expression of LAMTOR1 is abnormally high in nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC); hence, LAMTOR1 may play an important role in the development of NASH and HCC. Therefore, exploring the LAMTOR1 regulatory mechanism in the progression of NASH and malignant transformation of liver inflammation may be crucial for translational medicine. First, a NASH mouse model was established by feeding a methionine choline-deficient (MCD) diet. Hematoxylin-eosin staining of liver tissues showed successful modeling of inflammatory injury in the mouse liver. Immunoblot analysis confirmed LAMTOR1- and LAMTOR1-mediated protein expression in LAMTOR1 specifically depleted mouse livers. Subsequently, metabolic profiling of liver tissues was performed using an ultra-performance liquid chromatography-time-of-flight mass spectrometry strategy. Based on the retention time, m/z value, and tandem mass spectra, 134 metabolites were identified. Among these, the levels of 45 metabolite were significantly influenced by hepatic LAMTOR1 depletion. According to the metabolomics results, uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) was significantly upregulated in LAMTOR1-depleted (LAMTOR1LKO) hepatocyte tissues. As the final product of the hexosamine biosynthetic pathway (HBP), alteration in UDP-GlcNAc levels may regulate LAMTOR1 and metabolic regulatory genes downstream of HBP. Moreover, there was an obvious increase in the levels of several methylation-related metabolites. Thus, upregulated S-adenosylmethionine, S-adenosylhomocysteine, and N6,N6,N6-trimethyl-L-lysine indicated that LAMTOR1 may regulate the process of DNA or protein methylation. In addition, downregulation of 9-oxo-octadecadienoate, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) was also observed in LAMTOR1LKO mice liver tissues. Alterations in polyunsaturated fatty acids, such as EPA and DHA, link LAMTOR1 to inflammatory and immune processes, which are known to play important roles in NASH pathogenesis. These metabolic disorders demonstrated that LAMTOR1 significantly contributed to the metabolic mechanism of NASH. Furthermore, gene expression correlations were analyzed to interpret the regulatory role of LAMTOR1 from the perspective of genetic networks. Owing to a paucity of liver whole-transcriptome studies in NASH, correlation analysis was performed based on HCC transcriptome data from public databases. First, a negatively regulated relationship was observed between LAMTOR1 and MAT1A (R=-0.47). MAT1A encodes methionine adenosyltransferase 1A, an essential enzyme that catalyzes the formation of S-adenosylmethionine. Based on the upregulation of UDP-GlcNAc under hepatocyte LAMTOR1 depletion, it was predicted that LAMTOR1 positively influenced MGAT1 (R=0.47), a gene encoding alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase. Together with changes in succinyladenosine caused by hepatocyte LAMTOR1 deletion, predicted correlation results showed that LAMTOR1 may also participate in the pathogenesis through the positive regulatory relationship with ADSL (R=0.59). The ADSL gene provides instructions for making an enzyme called adenylosuccinate lyase, which can dephosphorylate the substrate succinyladenosine. In this study, LAMTOR1 was identified to specifically regulate multiple key metabolic pathways based on both NASH mouse models and gene expression correlations. These results illustrate the important role of LAMTOR1 in the progression of NASH and malignant transformation of liver inflammation, which provides a theoretical basis for the diagnosis and treatment of NASH or possible NASH-driven HCC.

Select

Preparation of dual-functional composite magnetic nanomaterials modified with different metals/aptamers and their performance in exosome enrichment ZHANG Weibing, LU Rui, ZHANG Lingyi 2021, 39 (10):  1128-1136.  DOI: 10.3724/SP.J.1123.2021.06012 Abstract ( 193 )   HTML ( 202 )   PDF (1442KB) ( 91 )   Supporting Information Exosomes, which are extracellular vesicles with sizes of 30-150 nm, contain proteins, lipids, RNA, etc., which can reflect important information about parental cells. They also have unique structures and can perform characteristic biological functions. Although the release of exosomes is a normal process, tumor cells release more exosomes, and the contents can induce cancer progression. Exosomes are widely distributed in body fluids at high concentrations and are easy to obtain; hence, the collection of exosomes released by tumor cells has become one of the main directions in tumor liquid biopsy. In order to ensure the reproducibility and consistency of liquid biopsy results, it is necessary to develop methods for enriching exosomes in sufficient yield and purity from complex samples. Based on the size, hydrophobic proteins, and characteristic proteins of exosomes, various methods for exosome separation and purification have been developed, such as ultracentrifugation, polymer precipitation, and immunoaffinity methods. An aptamer (Apt) is an oligonucleotide chain with a total length of 20-100 nt, which has ligand binding properties and can be used to detect different types of drugs and biomolecules at the nanomolar level. Characteristic proteins on the surface of exosomes such as CD63, CD9, and CD81 are often used as exosomes markers. At present, a variety of aptamer sequences targeting the characteristic proteins of exosomes have been reported. Zirconium and titanium cations as well as the oxides of these metals show high affinity to the phospholipid bilayer on the exosome surface and are used in the separation and purification of exosomes. Metal organic frameworks (MOFs) can provide a wealth of metal oxide affinity sites to interact with the phospholipid bilayer membrane, and their diverse organic ligands can provide numerous modification sites to bind with aptamers. In this study, different metal/aptamer dual-functional composite magnetic nanomaterials were prepared by exploiting the surface chemistry and biological characteristics of exosomes for the enrichment and purification of exosomes. Because of the specific affinity of the aptamers toward the target membrane protein on the exosome surface and the affinity of the titanium or zirconium oxide toward the phospholipid bilayer membrane of exosomes, dual-functional magnetic nanomaterials can greatly improve the enrichment capacity and separation selectivity of exosomes. Fe3O4@Zr-MOFs was used as the substrate to fabricate the dual functional MOFs/metal oxide aptamer composite magnetic nanomaterial Fe3O4@Zr-MOFs-Apt. UiO-66-NH2 was grown in situ on the surface of Fe3O4 by a solvothermal method to form a Zr-MOFs layer, and aptamer-CD63 was covalently bonded to the amino group of the organic ligand of the MOFs. The magnetic bimetallic metal organic framework Fe3O4@Zr-Ti-MOFs, which was fabricated via a layer-by-layer assembly approach, was used as the substrate to prepare the dual functional MOFs/metal oxide aptamer composite Fe3O4@Zr-Ti-MOFs-Apt via coordination bond formation between the metal site on the Fe3O4@Zr-Ti-MOFs and the aptamers. The third dual functional MOFs/metal oxide aptamer composite magnetic nanomaterial, Fe3O4@TiO2-Apt, was prepared by using Fe3O4@TiO2 as the substrate via coordination bond formation between the metal site on Fe3O4@TiO2 and the aptamers. Considering model exosomes extracted by ultracentrifugation and urine as samples, this paper compared the enrichment performance of materials modified with the same quality of aptamers and different levels of metal oxides. The dual-functional composite magnetic nanomaterials modified with different metals/aptamers were used for the enrichment of urine exosomes. The obtained exosomes were lysed and identified by mass spectrometry, and 233, 343, and 832 exosomal proteins were identified. This result also shows that dual-functional magnetic nanomaterials can fully combine the high selectivity of the nucleic acid aptamer and the high enrichment capacity of the metal oxides. The rapid, efficient separation and purification of exosomes in biological samples has excellent application potential. The material design and purification methods also provide a new idea for the development of new exosome-enrichment materials.

Select

Preparation of molecularly imprinted polymers-functionalized silica nanoparticles for the separation and recognition of aristolochic acids ZHANG Yuemei, GUO Lihua, LI Yijun, HE Xiwen, CHEN Langxing, ZHANG Yukui 2021, 39 (10):  1137-1145.  DOI: 10.3724/SP.J.1123.2021.06024 Abstract ( 291 )   HTML ( 210 )   PDF (4821KB) ( 144 )   Aristolochic acids (AAs), which is commonly found in Aristolochia and Asarum plants, has been widely used in several traditional medicine practices due to their anti-inflammatory, anti-malarial, and anti-hyperglycemic activities. Recently, researchers have found a “decisive link” between liver cancer and aristolochic acid after analyzing a large number of liver cancer samples around the world. Therefore, a highly sensitive and selective method is required for the analysis of AAs in traditional Chinese medicines (TCM). For the determination of AAs in TCM, pretreatment is indispensable because in actual TCM samples, AAs is present in trace amounts and the complex matrix exerts interference. In the past decades, molecularly imprinted polymers (MIPs) have attracted considerable attention as an alternative for the trace analysis in complicated matrices. In this study, MIP-coated SiO2 nanoparticles (SiO2@MIP NPs) was prepared for the determination of aristolochic acid by surface molecular imprinting using aristolochic acid Ⅰ (AAI ) as the template molecule, 2-vinylpyridine (VPY) as the functional monomer, and ethyleneglycol dimethacrylate (EGDMA) as the cross-linking agent. Core-shell-structure SiO2@MIP NPs were obtained by modifying vinyl groups on the surface of SiO2 NPs, coating MIPs films onto the silica surface via selective polymerization, and final extraction of template AAI and generation of the recognition site. To find a suitable functional monomer for the best imprinting effect, the interaction between the template and the functional monomers, including acrylic acid (AA), methyl acrylic acid (MAA), 2-vinyl pyridine (VPY), acrylamide (AM), and methylacrylamide (MAM) was investigated. Electrostatic interaction between AAI and VPY resulted in the maximum decrease in absorbance of AAI at 250 nm. Therefore, VPY was chosen for the preparation of MIP. The morphological and physical properties of the MIPs were characterized by transmission electron microscopy (TEM), Fourier-transform infrared (FT-IR) spectroscopy, thermogravimetric analysis, and N2 adsorption and desorption surface analysis. TEM images showed that SiO2 NPs were monodispersed with diameter of about 200 nm. The clear core-shell structure of SiO2@MIP NPs was observed, and the thickness of MIPs coating was about 35 nm. The FT-IR spectra of SiO2 NPs, vinyl group modified SiO2 and SiO2@MIP NPs revealed that the vinyl group and organic MIP layer were successfully modified at SiO2 sequentially. The results of thermogravimetric analysis were consistent with the FT-IR data for different SiO2 NPs. The nitrogen gas adsorption-desorption experiments showed that SiO2@MIP NPs and non-imprinted polymer (SiO2@NIP NPs) have the same pore volumes, while the surface area and pore size of MIPs were slightly larger than those of NIPs. Therefore, the difference in adsorption between SiO2@MIP NPs and SiO2@NIP NPs resulted from the imprinted sites on the MIP surface, rather than the difference in their surface areas. The adsorption properties of SiO2@MIP NPs were demonstrated by kinetic, isothermal, and selective adsorption experiments. The results of these experiments displayed that SiO2@MIP NPs reached adsorption equilibrium within a short period (120 s) and possessed a much higher rebinding ability than SiO2@NIP NPs. To verify the selectivity of SiO2@MIP NPs for AAI, three structural analogues (viz. tanshinone ⅡA, 2-methoxy-5-nitrophenol, and benzoic acid) were selected. The results showed that the binding capacity of SiO2@MIP NPs was much higher than those of these analogues. SiO2@MIP NPs have high adsorption capacity (5.74 mg/g), high imprinting factor (4.9), good selectivity coefficient (2.3-6.6) towards the structural analogues. SiO2@MIP NPs was used as an adsorbent and combined with HPLC for the selective separation of AAI in TCM. The recoveries of Kebia trifoliate samples spiked with three levels of AAI (0.3, 0.5, and 1.0 μg/mL) ranged from 73% to 83%. The results suggested that the proposed SiO2@MIP NPs could be used for selective enrichment of AAI from real complex TCM samples.