Offline two-dimensional liquid chromatography coupled with ion mobility-quadrupole time-of-flight mass spectrometry enabling fourdimensional separation and characterization of the multicomponents from white ginseng and red ginseng

Inherent complexity of plant metabolites necessitates the use of multi-dimensional information to accomplish comprehensive profiling and confirmative identification.A dimension-enhanced strategy,by offline two-dimensional liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry(2 D-LC/IM-QTOF-MS)enabling four-dimensional separations(2 D-LC,IM,and MS),is proposed.In combination with in-house database-driven automated peak annotation,this strategy was utilized to characterize ginsenosides simultaneously from white ginseng(WG)and red ginseng(RG).An offline 2 DLC system configuring an Xbridge Amide column and an HSS T3 column showed orthogonality 0.76 in the resolution of ginsenosides.Ginsenoside analysis was performed by data-independent high-definition MSE(HDMSE)in the negative ESI mode on a Vion?IMS-QTOF hybrid high-resolution mass spectrometer,which could better resolve ginsenosides than MSEand directly give the CCS information.An in-house ginsenoside database recording 504 known ginsenosides and 58 reference compounds,was established to assist the identification of ginsenosides.Streamlined workflows,by applying UNIFI?to automatedly annotate the HDMSEdata,were proposed.We could separate and characterize 323 ginsenosides(including 286 from WG and 306 from RG),and 125 thereof may have not been isolated from the Panax genus.The established 2 D-LC/IM-QTOF-HDMSEapproach could also act as a magnifier to probe differentiated components between WG and RG.Compared with conventional approaches,this dimensionenhanced strategy could better resolve coeluting herbal components and more efficiently,more reliably identify the multicomponents,which,we believe,offers more possibilities for the systematic exposure and confirmative identification of plant metabolites.

Dimension-Enhanced Ultra-High Performance Liquid Chromatography/Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry Combined with Intelligent Peak Annotation for the Rapid Characterization of the Multiple Components from Seeds of Descurainia sophia

The complex composition of herbal metabolites necessitates the development of powerful analytical techniques aimed to identify the bioactive components.The seeds of Descurainia sophia(SDS)are utilized in China as a cough and asthma relieving agent.Herein,a dimension-enhanced integral approach,by combining ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry(UHPLC/IMQTOF-MS)and intelligent peak annotation,was developed to rapidly characterize the multicomponents from SDS.Good chromatographic separation was achieved within 38 min on a UPLC CSH C18(2.1×100 mm,1.7μm)column which was eluted by 0.1%formic acid in water(water phase)and acetonitrile(organic phase).Collision-induced dissociation-MS^(2)data were acquired by the data-independent high-definition MS^(E)(HDMS^(E))in both the negative and positive electrospray ionization modes.A major components knockout strategy was applied to improve the characterization of those minor ingredients by enhancing the injection volume.Moreover,a self-built chemistry library was established,which could be matched by the UNIFI software enabling automatic peak annotation of the obtained HDMS^(E)data.As a result of applying the intelligent peak annotation workflows and further confirmation process,a total of 53 compounds were identified or tentatively characterized from the SDS,including 29 flavonoids,one uridine derivative,four glucosides,one lignin,one phenolic compound,and 17 others.Notably,four-dimensional information related to the structure(e.g.,retention time,collision cross section,MS^(1)and MS^(2)data)was obtained for each component by the developed integral approach,and the results would greatly benefit the quality control of SDS.

Screening and Identifying of Nephrotoxic Compounds in Lithospermum erythrorhizon Using Live-cell Fluorescence Imaging and Liquid Chromatography Coupled with Tandem Mass Spectrometry

In order to identify the potential nephrotoxic compounds in traditional Chinese medicine Lithospermum erythrorhizon,it was separated into serial fractions according to their polarities.An in vitro method was utilized to determine the nephrotoxicity of these fractions with the help of fluorescence image analysis.As a result,the primary fraction A05 and its secondary fractions C06 ＂C09 and C12 ＂C14 were found to have significant toxicity to LLC-PK1 cell line,as determined by the survive rate less than 20% after they were treated with these fractions.These potential nephrotoxic fractions were further analyzed by multistage and high resolution mass spectrometry.The main compounds in these fractions were tentatively identified to be acetylshikonin,isobutyrylshikonin,β,β＇-dimethyla-cryloylshikonin,and isovalerylshikonin,which may bring nephrotoxicity.

Simultaneous detection of 22 mycotoxins in grape by QuEChERS and ultra-high-performance liquid chromatography coupled with quadrupole/orbitrap high-resolution mass spectrometry

To effectively assess the extent of the mycotoxin contamination of grapes,a method was developed to analyze 22 mycotoxins by ultra-high-performance liquid chromatography coupled with quadrupole/orbitrap high-resolution mass spectrometry(UPLC-Q-Orbitrap HRMS).Samples were extracted and purified using the QuEChERS(quick,easy,cheap,effective,rugged,and safe)procedure.The extracts were separated on a BEH Shield C18 column(100 mm×2.1 mm,1.7μm),using methanol and water with 0.1%formic acid solution and 2 mmol/L ammonium acetate as the mobile phases.The quantification was performed according to a matrix-matched external standard method.There was a good linear relationship within the respective mass concentration ranges of 22 mycotoxins;the correlation coefficients were not less than 0.9981.The limit of quantification was 0.3-10.0μg/kg.Mycotoxin recovery ranged from 68.6%to 109.0%,with a relative standard deviation of 1.28%-12.80%.The method is simple,rapid,highly sensitive,and accurate,making it useful for screening grapes for common and emerging mycotoxins.

Ultra-performance Liquid Chromatography-Quadrupole/Time-of-Flight Mass Spectrometry Based Bile and Urine Metabonomics Study on the Ameliorative Effects of Curcuma wenyujin Rhizoma on Acute Blood Stasis in Rats

Background: Curcuma wenyujin rhizome(CWR) is a commonly used Chinese herbal medicine for treating blood stasis in China for 1000 of years. However, the underlying mechanism of CWR remains unclear. Aims and Objectives: The purpose of this study is to clarify the bioactive mechanism of CWR in treating blood stasis. Materials and Methods: In this study, pharmacological indexes, including hemorheology and four blood coagulation indexes were tested. Bile and urine metabolomics were engaged by UPLC-Q/TOF-MS. Multivariate statistical analysis were used to screen out differential endogenous metabolites. Results: The results indicated that CWR significantly ameliorated the hemorheology and coagulation functions of acute blood stasis(ABS) model rats. Moreover, 27 endogenous metabolites between the CWR group and the ABS group were screened, and the levels were all improved to certain degrees by CWR preadministration. Metabonomics results indicated that ABS was mainly related to linoleic acid metabolism, arachidonic acid metabolism, glycerophospholipid metabolism, sphingolipid metabolism, pentose and glucuronate intercereasonversions, steroid hormone biosynthesis, and primary bile acid biosynthesis. Conclusion: In a word, the metabolomics method is consistent with the holistic view of traditional Chinese medicine(TCM) that can be a powerful means to illustrate the biological activity mechanism of CWR in treating blood stasis and to offer research demonstration for further study on the effector mechanism of TCM.

COMPREHENSIVE TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY COUPLED WITH QUADRUPLE TIME-OF-FLIGHT MASS SPECTROMETRY FOR CHEMICAL CONSTITUENTS ANALYSIS OF TRIPTERYGIUM GLYCOSIDES TABLETS

Comprehensive two-dimensional liquid chromatography platform(LC×LC)coupled with quadrupole time-of-flight(QTOF)mass spectrometry(MS)is developed to separate,identify and relatively determine the chemical constituents of two types of tripterygium glycosides tablets(TGT).The types and relative contents of the constituents discovered in two kinds of TGT tablets were subsequently compared.C8andC18 column were used for the separation of the first

Rapid Differentiation of Aconiti Kusnezoffii Radix from Different Geographic Origins Using Ultra-Performance Liquid Chromatography Coupled with Time-of-Flight Mass Spectrometry

Objective:There are different geographic origins of Aconiti Kusnezoffii Radixs(AKRs)sold in the market with different quality.This study aims to establish a rapid analysis method to distinguish the different geographic origins of AKRs and to realize the rapid evaluation of their quality.Methods:An ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry(UPLC-Q-TOF MS)method was utilized to acquire the constituents'information of AKRs from different geographic origins.MSE data and Progenesis QI software were employed to identify the chemical constitutes.Principal component analysis(PCA)was applied to comparing MS data to find the chemical markers of AKRs from different geographic origins.Results:Twenty-three components were detected and 17 out of them were identified,including diester-diterpenoid alkaloids,monoester-diterpenoid alkaloids,and amine-diterpenoid alkaloids.Three pairs of isomers were detected and two of them were distinguished by the retention time of standard samples.Thirteen chemical markers were screened out through PCA and orthogonal partial least square discriminant analysis.Through detecting Napelline or isomer of Napelline(m/z 360.2530)and Aconifine(m/z 662.3170),AKRs from inner Mongolia autonomous could be screened.According to the existence of benzoylaconine(m/z 604.3108)and Indaconitine(m/z 630.3159),it could be confirmed that the AKRs are from Xinjiang Uygur autonomous.AKRs that cannot detect compounds above-mentioned could be from Liaoning or Shanxi Province.Conclusions:The chemical profile could be used not only to distinguish the AKRs from different geographic origins but also to identify the true and false of AKRs.This study lays a foundation for the study of efficacy and toxic of AKRs.

UPLC-QTOF/MS Method for Screening and Determination of 59 Non-labeled Components in Veterinary Drug Preparations

[Objectives]This study was conducted to provide an accurate and reliable method for rapid high-throughput screening of veterinary drug preparations.[Methods]For the matrixes of veterinary drug preparations,high-performance liquid chromatography-high resolution quadrupole time-of-flight mass spectrometry(UPLC-QTOF/MS)was used to establish a fast screening method for 59 non-standard components in five categories of antiviral agents,aminoglycosides,quinolones,sulfonamides,and tetracyclines in veterinary drugs.The target drugs were separated by a Waters ACQUITY UPLC HSS T3 chromatographic column(50 mm×2.1 mm,1.8μm),and data were collected in the positive ion mode.Good separation of the 59 drugs was achieved within 7 min.[Results]In the concentration range of 0-100 ng/ml,each drug showed a good linear relationship,and the correlation coefficients were all greater than 0.999.The detection limits of the 59 drugs were in the range of 0.1-0.5 mg/ml,and the recovery under the addition concentration of 5 mg/ml was in the range of 85.2%-103.8%.[Conclusions]The method is fast,simple,accurate,and highly sensitive,and is suitable for high-throughput screening and qualitative identification of non-standard components in veterinary drug preparations.

Application of UPLC-QTOF-MS Technology in Quality Stability Evaluation of Moluodan Concentrated Pills

[Objectives]To use liquid chromatography-mass spectrometry technology to analyze the chemical composition of traditional Chinese medicine and explore its application in the evaluation of quality stability of traditional Chinese medicine.[Methods]Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MS)was used to detect the samples of Moluodan concentrated pills.By comparing and analyzing the detection results of 10 different batches of Moluodan concentrated pills,combined with principal component analysis(PCA),the quality stability of Moluodan concentrated pills was evaluated.[Results]A total of 367 chemical components were identified in Moluodan concentrated pills.The average repetition rate of the chemical components contained in the 10 different batches of samples reached 92%.The overall quality stability of the Moluodan concentrated pills was good.[Conclusions]The UPLC-QTOF-MS technology combined with PCA provides a reference for the overall quality evaluation of Moluodan concentrated pills,and provides new detection methods and ideas for the analysis of the components of Chinese medicine.

Analysis of Pharmacodynamic Substance Basis of Fufang Changtai in Treating Colorectal Cancer by UPLC-Q-TOF-MS Combined with Network Pharmacology

[Objectives] To systematically study the main active components of Fufang Changtai(FFCT) in the treatment of colorectal cancer(CRC), and to explore its mechanism of action. [Methods] The main chemical components of FFCT were analyzed by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) combined with automatic analysis platform, and the main pharmacodynamic substances of FFCT were studied by network pharmacology method and its mechanism of action was explored. The binding degree between the active components and the core targets were verified by molecular docking technology. [Results] A total of 86 compounds were identified from FFCT, among which 26 compounds were Ginsenoside Rg3, Ginsenoside Rb1, Astragaloside III, etc. The key target pathway enrichment analysis showed that FFCT played its role in the treatment of CRC mainly through the PI3K-Akt signaling pathway and MAPK signaling pathway. [Conclusions] This study comprehensively identified the FFCT components. Supplemented by network pharmacology and molecular docking technology, it is expected to provide a scientific theoretical basis and an important reference for FFCT therapeutic components identification, key target verification and mechanism of action in the treatment of CRC.

Using cell membrane chromatography and HPLC-TOF/MS method for in vivo study of active components from roots of Aconitum carmichaeli

An offline two-dimensional system combining a rat cardiac mascle cell membrane chromatography time-of-flight mass spectrometry （CMC-TOF/MS） with a high performance liquid chromatography time-of-flight mass spectrometry （HPLC-TOF/MS） was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli. On the basis of the analysis of the first dimension, retention components of the urine sample were collected into 30 fractions （one fraction per minute）. Then offline analysis of the second dimension was carried out. 34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine, and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method. As a result, binding affinities decreased along with the process of deacylation, debenzoylation and demethylation, which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism. Moreover, some minor components in rat urine （Songorine, 14-benzoylneoline, Deoxyaconitine, etc. ） exerted relatively strong affinity on cell membranes are worth exploring. The results delivered by the system suggest that the CMC can be applied to in vivo study.

液相色谱/四极杆-飞行时间质谱测定失忆性贝毒的研究

失忆性贝毒(ASP)是一种80年代在加拿大新发现的毒素,是由大量Pseudonitzschia硅藻属海藻生长产生的软骨藻酸(domoic acid,DA,图1)污染所致,与产生ASP相关的贝类包括贻贝等.……

Serum metabolic profiling of targeted bile acids reveals potentially novel biomarkers for primary biliary cholangitis and autoimmune hepatitis

BACKGROUND Primary biliary cholangitis(PBC)and autoimmune hepatitis(AIH)are two unexplained immune diseases.The golden standard for diagnosis of these diseases requires a liver biopsy.Liver biopsy is not widely accepted by patients because of its invasive nature,and atypical liver histology can confuse diagnosis.In view of the lack of effective diagnostic markers for PBC and AIH,combined with the increasingly mature metabolomics technologies,including full-contour metabolomics and target.AIM To determine non-invasive,reliable,and sensitive biochemical markers for the differential diagnosis of PBC and AIH.METHODS Serum samples from 54 patients with PBC,26 patients with AIH and 30 healthy controls were analyzed by Ultra-high performance liquid chromatographytandem mass spectrometry serum metabolomics.The metabolites and metabolic pathways were identified,and the metabolic changes,metabolic pathways and inter-group differences between PBC and AIH were analyzed.Fifteen kinds of target metabolites of bile acids(BAs)were quantitatively analyzed by SRM,and the differential metabolites related to the diagnosis of PBC were screened by receiver operating characteristic curve analysis.RESULTS We found the changes in the levels of amino acids,BAs,organic acids,phospholipids,choline,sugar,and sugar alcohols in patients with PBC and AIH.Furthermore,the SRM assay of BAs revealed the increased levels of chenodeoxycholic acid,lithocholic acid(LCA),taurolithocholic acid(TLCA),and LCA+TLCA in the PBC group compared with those in the AIH group.The levels of BAs may be used as biomarkers to differentiate PBC from AIH diseases.The levels of glycochenodeoxycholic acid,glycochenodeoxycholic sulfate,and taurodeoxycholic acid were gradually elevated with the increase of Child-Pugh class,which was correlated with the severity of disease.CONCLUSION The results demonstrated that the levels of BAs could serve as potential biomarkers for the early diagnosis and assessment of the severity of PBC and AIH.

Bioprospecting optimal phenology for bioactive molecules in native golden yellow Pleurotus citrinopileatus Singer

Objective: To bioprospect optimal phenological phases as source of novel molecules from native golden yellow Pleurotus citrinopileatus across four phenologies in both aqueous and ethanol extracts, and identify novel molecules responsible for these activities.Methods: Standard qualitative assay, Folin–Ciocalteu assay; aluminium chloride spectrophotometric, 2, 2-diphenyl-1-picrylhydrazyl, 2, 20-azinobis(3-ethylbenzothiazoline-6-suslfonic acid, ferricyanide reducing antioxidant power were used to determine total flavonoid, polyphenols, radical scavenging, and reducing power. Spectrophotometric methods were used for lycopene, b-carotene, and total carotenoids, while liquid chromatography quadrupole time of flight mass spectrometry was used for identification and comparative quantitation of polyphenols and flavonoids across the four phenological states. Chem Spider? database was used for the identification of compounds based on their empirical formula, accurate mass and literature review of previously reported compounds in mushroom.Results: Primordial phases exhibited higher contents of secondary metabolites than mature basidiocarps. Polyphenols content differed across physiological phases with primordials exhibiting significant high contents(P < 0.05) [(13.803 ± 0.797) mg gallic acid equivalent/g dry weight]. Distribution of total flavonoids was significantly different(P < 0.05) across physiological states and ranged from(3.311 ± 0.730) to(14.824 ± 0.890) mg quercetin equivalent g dry weight. Ten polyphenol acids and seven flavonoids compounds identified varied across these phases with primordials exhibiting relatively high peak areas. Total antioxidant activities showed a positive correlation with total polyphenols(r = 0.969;P < 0.05) and total flavonoids(r = 0.960; P < 0.05) across these phenologies.Conclusions: These findings provide evidence that primordials of golden yellow mushroom as opposed to their fruiting bodies are potent sources of bioactive health molecules.

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.

Rapid Profiling and Characterization of the Multicomponents from the Root and Rhizome of Salvia miltiorrhiza by Ultra-High Performance Liquid Chromatography/Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry in Combination with Computational Peak Annotation Workflows

Herbal components characterization represents a challenging task because of the co-existing of multiple classes of naturally occurring compounds with wide spans of polarity,molecular mass,and the ubiquitous isomerism.The root and rhizome of Salvia miltiorrhiza have been utilized as a reputable traditional Chinese medicine Salviae Miltiorrhizae Radix et Rhizoma(Dan-Shen)in the treatment of cardiovascular disease.Herein,a dimensionenhanced ultra-high performance liquid chromatography/ion mobility/quadrupole time-of-flight mass spectrometry approach in combination with intelligent peak annotation workflows was established aimed to rapidly characterize the multicomponents from S.miltiorrhiza.Due to the sufficient optimization,satisfactory chromatography separation was enabled on an HSS T3 column within 33 min using 0.1%formic acid in water(A)and acetonitrile(B)as the mobile phase,while the data-independent HDMS^(E) in both the negative and positive electrospray ionization modes was utilized for the high-coverage MS^(2) data acquisition.Streamlined automatic peak annotation by searching an in-house library(recording 198 known compounds)followed by the subsequent confirming steps(e.g.,comparison with the reference compounds,fragmentation pathways analysis,and retention behavior comparison,etc.),allowed us to identify or tentatively characterize a total of 86 components(including 50 terpenoids,21 phenolic acids,and 15 others)from S.miltiorrhiza.Importantly,three-dimensional structure information,such as the retention time,MS^(1) and MS^(2) data,and collision cross section(CCS),was provided,which can facilitate the more reliable characterization of herbal components.

Isolation and purification of uremic middle molecules by multi-step liquid chromatography

Isolation and comparison of uremic sera and urine and normal sera and urine were performed by gel permeation chromatography, anion exchange chromatography and re-versed-phase high performance liquid chromatography. Two uremic middle molecular fractions (A and B) were obtained from uremic sera and urine and normal urine by gel permeation chromatography, but not from normal sera. The anion exchange chromatographic results of fraction A from different origins demonstrate that subfraction A-3 could be excreted in urine by healthy subject, but accumulated in uremic serum for renal failure of patient with uremia. After desalinization subfraction A-3 was analyzed by MALDI-TOF-MS. The results show that subfraction A-3 consists of six compounds with molecular weight 839, 873, 1007.94, 1106, 1680 and 2015 respectively. Finally, by reversed-phase high performance liquid chromatography, subfraction A-3 was further resolved into six independent fractions. Thus, the isolation and purification of six middle molecular

地衣内生真菌Pestalotiopsis sp.次生代谢产物

拟盘多毛孢属Pestalotiopsis真菌由于能够产生大量结构新颖活性显著的次生代谢产物,特别是从中发现抗肿瘤药物紫杉醇,成为近年来研究的热点。本研究通过对1株地衣内生真菌Pestalotiopsis sp.的固体培养基次生代谢产物的UPLC-Q-TOF-MS分析,发现该菌株能够产生分子量比较特殊的两个色谱峰。通过进一步的菌株大量发酵,结合硅胶柱色谱,高效液相色谱等技术跟踪分离,得到了2个单体化合物。采用核磁共振波谱技术、质谱技术等方法确定这2个单体化合物平面结构,分别为已知torrenyanic acid衍生物(1)和新化合物pestalotiopsin(2)。比对化合物的CD谱及考虑生源关系,确定了新化合物(2)的绝对构型。化合物1和2的体外抑制人白血病细胞K562实验显示二者均具有抗肿瘤活性,IC50值分别为25.2和32.1μmol/L。

Characterization and quantitation of aristolochic acid analogs in different parts of Aristolochiae Fructus, using UHPLC-Q/TOF-MS and UHPLC-Qq Q-MS

Aristolochiae Fructus, a Chinese herbal medicine derived from the fruit of Aristolochia contorta Bge., contains nephrotoxic aristolochic acid analogues(AAAs). According to ancient medical texts, various medicinal parts of the fruit of A. contorta were ever used. In order to reveal which part could be safely and effectively used, it is necessary to analyze the chemical profiles of different medicinal parts. Herein we compared the chemical compositions and determined aristolochic acid I(AA-I) and aristolochic acid II(AA-II) in the four parts viz. outer pericarp, inner pericarp, septum, and seed. Ultra-high performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry(UHPLC-QTOF-MS) was applied for chemical profiling. Ultra-high performance liquid coupled with triple quadrupole mass spectrometry(UHPLC-Qq Q-MS) was employed to quantify AA-I and AA-II in different parts. It was found that the chemical compositions of the four parts varied both qualitatively and quantitatively. A total of 10 AAAs, including 5 aristolochic acids and 5 aristolactams, together with 3 alkaloids, were unambiguously or tentatively identified by UHPLC-QTOF-MS. The quantitatively analytical results obtained by UHPLC-Qq Q-MS showed that AA-I and AA-II exclusively accumulate in the seeds of A. contorta. These findings provide supporting data for the rational selection of medicinal parts.

Identification of Biomarkers for Endometriosis Using Clinical Proteomics

Background：We investigated possible biomarkers for endometriosis （EM） using the ClinProt technique and proteomics methods.Methods：We enrolled 50 patients with EM,34 with benign ovarian neoplasms and 40 healthy volunteers in this study.Serum proteomic spectra were generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MS） combined with weak cationic exchange （WCX） magnetic beads.Possible biomarkers were analyzed by a random and repeat pattern model-validation method that we designed,and ClinProtools software,results were refined using online liquid chromatography-tandem MS.Results：We found a cluster of 5 peptides （4210,5264,2660,5635,and 5904 Da）,using 3 peptides （4210,5904,2660 Da） to discriminate EM patients from healthy volunteers,with 96.67％ sensitivity and 100％ specificity.We selected 4210 and 5904 m/z,which differed most between patients with EM and controls,and identified them as fragments of ATP1B4,and the fibrinogen alpha （FGA） isoform 1/2 of the FGA chain precursor,respectively.Conclusions：ClinProt can identify EM biomarkers,which-most notably-distinguish even early-stage or minimal disease.We found 5 stable peaks at 4210,5264,2660,5635,and 5904 Da as potential EM biomarkers,the strongest of which were associated with ATP1B4 （4210 Da） and FGA （5904 Da）; this indicates that ATP1B4 and FGA are associated with EM pathogenesis.