Identification of endogenous carbonyl steroids in human serum by chemical derivatization, hydrogen/deuterium exchange mass spectrometry and the quantitative structure-retention relationship.

Steroids are tetracyclic aliphatic compounds, and most of them contain carbonyl groups. The disordered homeostasis of steroids is closely related to the occurrence and progression of various diseases. Due to high structural similarity, low concentrations in vivo, poor ionization efficiency, and interference from endogenous substances, it is very challenging to comprehensively and unambiguously identify endogenous steroids in biological matrix. Herein, an integrated strategy was developed for the characterization of endogenous steroids in serum based on chemical derivatization, ultra-performance liquid chromatography quadrupole Exactive mass spectrometry (UPLC-Q-Exactive-MS/MS), hydrogen/deuterium (H/D) exchange, and a quantitative structure-retention relationship (QSRR) model. To enhance the mass spectrometry (MS) response of carbonyl steroids, the ketonic carbonyl group was derivatized by Girard T (GT). Firstly, the fragmentation rules of derivatized carbonyl steroid standards by GT were summarized. Then, carbonyl steroids in serum were derivatized by GT and identified based on the fragmentation rules or by comparing retention time and MS/MS spectra with those of standards. H/D exchange MS was utilized to distinguish derivatized steroid isomers for the first time. Finally, a QSRR model was constructed to predict the retention time of the unknown steroid derivatives. With this strategy, 93 carbonyl steroids were identified from human serum, and 30 of them were determined to be dicarbonyl steroids by the charge number of characteristic ions and the number of exchangeable hrdrogen or comparing with standards. The QSRR model built by the machine learning algorithms has an excellent regression correlation, thus the accurate structures of 14 carbonyl steroids were determined, among which three steroids were reported for the first time in human serum. This study provides a new analytical method for the comprehensive and reliable identification of carbonyl steroids in biological matrix.

Qualitative Distribution of Endogenous Cholesteryl Esters in Plasma of Humans and Three Rodent Species Using Stepwise UPLC-Q-Exactive-MS.

OBJECTIVE: Cholesteryl esters (CEs) are composed of various fatty acyl chains attached to the hydroxyl groups of cholesterol, and abnormalities in their metabolism are related to many diseases. This study aimed to develop an ultrahigh-performance liquid chromatography-quadrupole exactive mass spectrometry (UPLC-Q-Exactive MS) method to identify the CEs in plasma. METHODS: First, the MS fragmentation patterns were investigated using seven commercial CE standards. Then, the CEs in plasma were characterized through the accurate mass data of precursor ions and characteristic product ions. A strategy of step-by-step m/z scans in a narrow range was proposed to identify more trace CEs by the full-scan data-dependent MS/MS (ddMS2) mode. RESULTS: A total of 50 CE species consisting of 55 regioisomers were identified in human plasma. Among them, two species were reported for the first time. CONCLUSION: This study is the most comprehensive identification of CE species in human plasma to date. These results will contribute to the in-depth profiling of CEs in human plasma and provide guidance for animal model selection when studying lipid-related diseases.

Experimental and theoretical study on the propagation characteristics of stress wave in filled jointed rock mass.

This study is to theoretically and experimentally investigate the propagation of stress waves in the filled joint set. The time-domain recursive method is used to derive the propagation equations in the filled joint set, and the filled joints are further simplified into structural planes without joint thickness. The split-Hopkinson rock bar is modified to simulate P wave propagation normally across the parallel filled joints. The relationship among stress-closure curve, joint specific stiffness, transmission coefficient and loading rate is analyzed. The results show that, for the rock mass with a single joint, both the joint specific stiffness and transmission coefficient of different filling materials increase with loading rate. More serious particle breakage of the filling materials leads to lower joint specific stiffness and transmission coefficient. For the rock mass with two joints, the joint specific stiffness of each joint affects the transmission coefficient of the filled joint set. It is found that our theoretical calculations are basically consistent with the experimental ones, and the joint specific stiffness can well characterize the propagation behavior of stress wave in the filled parallel rock joints.

Quantitative proteomics of plasma and liver reveals the mechanism of turmeric in preventing hyperlipidemia in mice.

Hyperlipidemia is manifested by abnormal levels of circulating lipids and may lead to various cardiovascular diseases. Studies have demonstrated that turmeric supplemented in food can effectively prevent hyperlipidemia. The aim of this study is to elucidate the underlying mechanism. 27 male C57BL/6J mice were randomly divided into three groups, which were fed with a standard diet, a high-fat diet and a high-fat diet supplemented with turmeric powder (2.0% w/w), respectively. After eight weeks of feeding, turmeric intervention significantly reduced the plasma TC, TG, and LDL-C levels and the LDL-C/HDL-C ratio of mice compared with high-fat diet fed mice. TMT-based proteomic analysis showed that the expression of 24 proteins in mouse plasma and 76 proteins in mouse liver was significantly altered by turmeric, respectively. Bioinformatics analysis showed that differential proteins in the plasma were mainly involved in complement and coagulation cascades and the cholesterol metabolism pathway. The differential proteins in the liver were mainly involved in arachidonic acid metabolism, steroid hormone biosynthesis and the PPAR signaling pathway. Key differential proteins were successfully validated by western blot analysis. This study is the first to reveal the preventive mechanism of turmeric on hyperlipidemia from proteomics. The results showed that dietary turmeric could prevent hyperlipidemia through regulating the expression of proteins in metabolism pathways.

Systematic Identification of Proteins Binding with Cisplatin in Blood by Affinity Chromatography and a Four-Dimensional Proteomic Method.

Cisplatin is widely used for the treatment of various solid tumors. It is mainly administered by intravenous injection, and a substantial amount of the drug will bind to plasma proteins, a feature that is closely related to its pharmacokinetics, activity, toxicity, and side effects. However, due to the unique properties of platinum complexes and the complexity of the blood proteome, existing methods cannot systematically identify the binding proteome of cisplatin in blood. In this study, high-abundance protein separation and an ion mobility mass spectrometry-based 4D proteomic method were combined to systematically and comprehensively identify the binding proteins of cisplatin in blood. The characteristic isotope patterns of platinated peptides and a similarity algorithm were utilized to eliminate false-positive identification. Finally, 39 proteins were found to be platinated. Bioinformatics analysis showed that the identified proteins were mainly involved in the complement and coagulation cascade pathways. The binding ratio of some peptides with cisplatin was measured based on the area ratio of the free peptide using the parallel reaction monitoring method. This study provides a new method for systematically identifying binding proteins of metal drugs in blood, and the identified proteins might be helpful for understanding the toxicity of platinum anticancer drugs.

Qualitative distribution of endogenous sphingolipids in plasma of human and rodent species by UPLC-Q-Exactive-MS.

Sphingolipids (SLs) are endogenously bioactive molecules with diverse structures, and its metabolic disorders are involved in the progression of many diseases. In this study, an ultra-performance liquid chromatography quadrupole exactive mass spectrometry (UPLC-Q-Exactive-MS) method was established to comprehensively profile SLs in plasma. First, the fragment patterns of SL standards of each subclass were investigated. Then, the SL species in plasma were characterized based on the fragmentation rules. Finally, a total of 144 endogenous SL species consisting of 216 regioisomers were identified in plasma of human, golden hamster and C57BL/6 mice, which was the most comprehensive identification for SLs in plasma. In addition to the known species, 19 SL species that have never been reported were also identified. The profile of SLs in plasma of human and two rodent species was compared subsequently. It was worth noting that a total of 9 SL molecular species consisting of 11 regioisomers with low abundance were successfully identified in human plasma through comparison among species. Those findings contribute to a deeper understanding of SLs in human plasma and provide scientific basis for the selection of animal model. The established profile of SLs in plasma could be used for screening of lipid biomarkers of various diseases.

The Protein-Binding Behavior of Platinum Anticancer Drugs in Blood Revealed by Mass Spectrometry.

Cisplatin and its analogues are widely used as chemotherapeutic agents in clinical practice. After being intravenously administrated, a substantial amount of platinum will bind with proteins in the blood. This binding is vital for the transport, distribution, and metabolism of drugs; however, toxicity can also occur from the irreversible binding between biologically active proteins and platinum drugs. Therefore, it is very important to study the protein-binding behavior of platinum drugs in blood. This review summarizes mass spectrometry-based strategies to identify and quantitate the proteins binding with platinum anticancer drugs in blood, such as offline high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC-ICP-MS) combined with electrospray ionization mass spectrometry (ESI-MS/MS) and multidimensional LC-ESI-MS/MS. The identification of in vivo targets in blood cannot be accomplished without first studying the protein-binding behavior of platinum drugs in vitro; therefore, relevant studies are also summarized. This knowledge will further our understanding of the pharmacokinetics and toxicity of platinum anticancer drugs, and it will be beneficial for the rational design of metal-based anticancer drugs.

Discrimination of turmeric from different origins in China by MRM-based curcuminoid profiling and multivariate analysis.

In this research, a three-step strategy was utilized for discriminating turmeric samples from different provinces and regions in China. Firstly, MRM-based UPLC-MS/MS method for chemical profiling of curcuminoids in turmeric samples was established. Then, response surface methodology was applied for optimizing the extraction process of targeted curcuminoids. Finally, multivariate analysis was conducted for systematic characterization of 66 curcuminoids in turmeric. Principal component analysis (PCA) and orthogonal projection to latent structure-discriminant analysis (OPLS-DA) revealed that turmeric samples from Sichuan and other regions could be classified into two distinct groups. Turmeric samples from the same group had similar curcuminoids content distribution. 25 differential curcuminoids were discovered through OPLS-DA, among which most curcuminoids were more abundant in Sichuan. Furthermore, turmeric samples from different provinces could be clearly discriminated based on hierarchical cluster analysis (HCA) using the screened differential curcuminoids.

Qualitative distribution of endogenous phosphatidylcholine and sphingomyelin in serum using LC-MS/MS based profiling.

PCs and SMs are the major types of glycerophospholipids and sphingophospholipids, the two main categories of phospholipids (PLs). To study the qualitative distribution of serum phosphatidylcholine (PC) and sphingomyelin (SM) in human and three rodent species, liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap-MS/MS) was used to identify them comprehensively through the accurate mass measurement of both precursor ions and their corresponding product ions. Based on the fragmentation rules of standards, the product ions at m/z 184.0733 were filtered to maximally screen possible PC and SM molecules. For PC, the fatty acid at sn-1 and sn-2 of the glycerol backbone was identified based on the product ions in negative mode. A total of 91 PCs and 31 SMs molecular species, consisting of 166 PCs and 39 SMs regioisomers, were detected in human serum, which is the most comprehensive identification of PC and SM species in serum. The qualitative distributions of PC in rat and SM in golden hamster, respectively, were more similar with that of human from an overall perspective. Those results provided guidance regarding to the animal model selection for mimicking lipid related-syndromes or diseases in human.

Spectrum-effect relationship for anti-tumor activity of shikonins and shikonofurans in medicinal Zicao by UHPLC-MS/MS and chemometric approaches.

Shikonin, shikonofuran and their derivatives are the main bioactive components of Zicao, a traditional Chinese medicine prepared with the dried roots of Lithospermum erythrorhizon, Arnebia euchroma or Arnebia guttata. To establish an efficient and sensitive method for studying material basis of Zicao, different scan modes of ultra-high performance liquid chromatography quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) and UHPLC triple quadrupole linear ion trap mass spectrometry (QTRAP-MS/MS) were incorporated to make full use of the sensitivity of multiple reaction monitoring (MRM) and overcome its disadvantages. A total of 73 shikonins and shikonofurans compounds were detected in Zicao utilizing various scanning modes. Thereafter the characteristic chemical profile for shikonins and shikonofurans was established based on UHPLC-QTRAP-MS/MS, which was subsequently used to study the spectrum-effect relationship by correlating the relative quantity of compounds and the anti-tumor activity. As a result, 27 compounds were screened as the main active components inhibiting HeLa cells by othogonal partial least square (OPLS). Among them, shikonin, acetylshikonin have been reported to inhibit HeLa cells previously, and ß, ß-dimethylacrylshikonin has been reported to be active component by other method. Those results showed that chemical characteristic profile combined with chemometric methods was efficient and reliable for discovery of material basis in TCM, especially trace active compounds.

Identification of the lipid-lowering component of triterpenes from Alismatis rhizoma based on the MRM-based characteristic chemical profiles and support vector machine model.

It has been demonstrated that triterpenes in Alismatis rhizoma (Zexie in Chinese, ZX) contributed to the lipid-lowering effect on high-fat diet-induced hyperlipidemia. Alisol B 23-acetate, one of the abundant triterpenes in ZX, was used as the marker of quality control for ZX in Chinese Pharmacopoeia, while it could not reflect the lipid-lowering effect because other triterpenes in ZX also had prominent medicinal efficacy. To identify the significantly bioactive triterpenes in ZX, a multiple reaction monitoring (MRM)-based characteristic chemical profile (CCP)-support vector machine (SVM) model was used to explore the relationship between triterpenes and lipid-lowering effect of ZX. Firstly, the content of 87 targeted triterpenes was quantified by the MRM-based CCP using UHPLC-QTRAP-MS/MS. Secondly, the lipid-lowering effect of 30 ZX samples was assessed by 3T3-L1 preadipocytes. Thirdly, 9 of the 87 triterpenes possessing high mean impact value were identified to have significant lipid-lowering effect via the particle swarm-optimized SVM model. The new SVM model constructed by the 9 triterpenes showed good prediction performance and the overall prediction accuracy reached 81.94%. Finally, the real activity of these triterpenes was partly confirmed and was consistent with the prediction of SVM. These results showed that the method for discovery of triterpenes with prominent lipid-lowering activity in ZX was reliable. The proposed method is expected to provide an efficient and rapid approach for screening of active component and drug discovery in traditional herbs. Graphical abstract.

Elucidation of the Mechanism of Action for Metal Based Anticancer Drugs by Mass Spectrometry-Based Quantitative Proteomics.

The discovery of the anticancer activity of cisplatin and its clinical application has opened a new field for studying metal-coordinated anticancer drugs. Metal-based anticancer drugs, such as cisplatin, can be transported to cells after entering into the human body and form metal⁻DNA or metal⁻protein adducts. Then, responding proteins will recognize adducts and form stable complexes. The proteins that were binding with metal-based anticancer drugs were relevant to their mechanism of action. Herein, investigation of the recognition between metal-based anticancer drugs and its binding partners will further our understanding about the pharmacology of cytotoxic anticancer drugs and help optimize the structure of anticancer drugs. The "soft" ionization mass spectrometric methods have many advantages such as high sensitivity and low sample consumption, which are suitable for the analyses of complex biological samples. Thus, MS has become a powerful tool for the identification of proteins binding or responding to metal-based anticancer drugs. In this review, we focused on the mass spectrometry-based quantitative strategy for the identification of proteins specifically responding or binding to metal-based anticancer drugs, ultimately elucidating their mechanism of action.

The strategy for establishment of the multiple reaction monitoring based characteristic chemical profile of triterpenes in Alismatis rhizoma using two combined tandem mass spectrometers.

It was reported that triterpenes compounds in Alismatis rhizoma (AR) contributed to the lipid lowering effect on high fat diet (HFD)-induced hyperlipidemia. To date only 24 triterpenes (including the isomers) were characterized by LC-QTOF-MS/MS due to the lack of strategies for systematic discovery, classification and identification of triterpenes in AR. In this study, an integrated strategy combining various QTOF-MS/MS and QTRAP-MS/MS scan functions was developed for systematic identification and specific characterization of triterpenes in AR and processed AR. First, MS/MS fragmentation behaviors of different types of triterpenes were investigated and their diagnostic product ions were systematically summarized for discovery and classification of triterpenes. Second, diagnostic product ions were used to filter the data acquired by UHPLC-QTOF MS/MS for efficient identification of targeted triterpenes. Third, MRM-based characteristic chemical profile (CCP) of triterpenes was established using 30 MRM transitions by UHPLC-QTRAP-MS/MS. Fourth, MRM-based CCP was applied for comparative analyses of triterpenes in AR from different regions and from two other processed AR (salt processed AR and bran processed AR). Consequently, a total of 80 triterpenes including 14 novel compounds were identified in the AR, and 7 more triterpenes compounds were discovered using MRM-based CCP in the processed AR. This work is the most comprehensive characterization of triterpenes compounds in AR to date. The established MRM-based CCP of triterpenes compounds can be instructive for qualitative analyses and relative quantitative analyses of triterpenes in AR and its related medicinal products for potential applications including quality control and classification of different AR materials.

Identification and characterization of curcuminoids in turmeric using ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry.

A three-step strategy was developed for systematic characterization of curcuminoids in turmeric. Based on UHPLC-QTOF-MS/MS analysis, 89 curcuminoids including 16 novel ones were identified in the turmeric samples using this approach. During the identification process, false positive results were excluded by combining the positive and negative ESI-MS/MS analyses. Moreover, the characterization of the keto and enol forms of type A, B and C curcuminoids was first discussed and they were clearly distinguished using negative ESI-MS/MS method with UV spectra analyses. The structures of detected curcuminoids were identified and rationalized in both ion modes. Additionally, the fragmentation behaviors of the 15 types of curcuminoids were clearly illustrated in this work, which will be helpful for detection and identification of corresponding trace curcuminoids in complex turmeric samples using UHPLC-QTOF-MS/MS methods.

An integrated strategy for establishment of curcuminoid profile in turmeric using two LC-MS/MS platforms.

Turmeric and curcuminoids are used as natural food coloring and functional food additives in various parts of the world. In this study, ninety-six curcuminoids were fully characterized using a targeted curcuminoid profile, which established by integrated use of two complementary LC-MS/MS platforms (liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOF-MS/MS) and liquid chromatography-quadrupole linear ion trap mass spectrometry (LC-QTRAP-MS/MS)). The curcuminoid profile was represented in the form of a multiple reaction monitoring (MRM) mode based on LC-QTRAP-MS/MS analysis. It facilitated the qualitative and relative quantitative analysis of curcuminoids in a single injection. Meanwhile, the profile was successfully applied to the quality evaluation of raw materials of turmeric from different regions in China and Myanmar. The structural identification procedures of curcuminoids and the integrated strategy provide a suitable method to analyze targeted plant metabolites which occur in a high number but sharing either structural similarities or similar functional groups.

The Hypolipidemic Effect of Total Saponins from Kuding Tea in High-Fat Diet-Induced Hyperlipidemic Mice and Its Composition Characterized by UPLC-QTOF-MS/MS.

Kuding tea are used as a traditional tea material and widely consumed in China. In this study, total saponins (TS) from water extract of Kuding tea was prepared by D101 macroporous resins and analyzed by UPLC-QTOF-MS/MS. Then the hypolipidemic effect of TS extract was investigated in high-fat diet-induced hyperlipidemic mice. For comprehensive identification or characterization of saponins in TS extract, 3 major saponins of Kudinoside A, Kudinoside F, and Kudinoside D were isolated and used as standards to investigate the MS/MS fragmentation pattern. As a result, 52 saponins were identified or characterized in TS extract from Kuding tea. In addition, the increased levels of mice serum TC, LDL-C, HDL-C, and atherogenic index (AI) were significantly reduced after the treatment of TS extract. Also, the liver protective effect of TS extract was obviously judged from the photographs stained with oil red-O staining. Meanwhile, TS extract significantly upregulated the expression of hepatic scavenger receptors including SR-AI, SR-BI, and CD36. Therefore, it is reasonable to assume that the overexpression of hepatic scavenger receptors was involved in the hypolipidemic effect of Kuding tea on the high-fat diet-induced hyperlipidemic mice. The TS extract could influence these scavenger receptors, and this could be the potential mechanism of TS extract from Kuding tea in the treatment of lipid disorders. These results give the evidence that the saponins in Kuding tea could provide benefits in managing hypercholesterolemia and may be a good candidate for development as a functional food and nutraceutical.