Two-dimensional gel electrophoresis and surface-enhanced laser desorption ionization-time of flight-mass spectrometry for detection of protein expression profiles in the hippocampus following closed brain injury

Gene expression profile changes in brain regions following traumatic brain injury at the gene level cannot sufficiently elucidate gene expression time, expression amount, protein post-translational processing or modification. Therefore, it is necessary to quantitatively analyze the gene expression profile using proteomic techniques. In the present study, we established a rat model of closed brain injury using Marmarou＇s weight-drop device, and investigated hippocampal differential protein expression using two-dimensional gel electrophoresis and surface-enhanced laser desorption ionization-time of flight-mass spectrometry. A total of 364 protein peaks were detected on weak cation exchange-2 protein chips, including 37 differential protein peaks. 345 protein peaks were detected on immobilized metal affinity capture arrays-Cu, including 12 differential protein peaks Further examination of these differential proteins revealed that glucose-regulated protein and proteasome subunit alpha type 3 expression were significantly upregulated post-injury. These results indicate that brain injury can alter protein expression in the hippocampus, and that glucose-regulated protein and proteasome subunit alpha type 3 are closely associated with the occurrence and development of traumatic brain injury.

Cerebrospinal fluid diagnostic markers for two-dimensional electrophoresis-mass spectrometry in Parkinson’s disease patients

BACKGROUND： Previous studies have confirmed the existence of specific proteins in body fluid of Parkinson＇s disease （PD） patients. However, the existing research has contained several interference factors with poor reproducibility and has not focused on patients grouped according to disease duration. OBJECTIVE： To verify differential expression of proteins in cerebrospinal fluid of PD patients grouped in order of disease severity through the use of two-dimensional electrophoresis-mass spectrometry methods. DESIGN, TIME AND SETTING： The proteomic-based, case-control study was performed between September 2008 and June 2009 at the Key Laboratory of Neurology in the First Affiliated Hospital of Chongqing Medical University. PARTICIPANTS： A total of 52 outpatients and/or inpatients, who were admitted to the Department of Neurology in the First Affiliated Hospital of Chongqing Medical University between 2008 and 2009, were randomized into the present study. Among them, 27 PD patients served as the PD group and were assigned to three subgroups according to modified Webster, Hoehn, and Yahr rating scales： 14 = mild, 8 = moderate, and 5 = severe; non-PD group of 16 patients included 5 cases of viral meningitis, 3 cases of acute myelitis, 1 case of Guillain-Barre syndrome, 2 cases of tuberculous meningitis, 2 cases of restless legs syndrome, and 3 cases of essential tremor; control group （n = 9） consisted of muscular tension headache in 6 cases, as well as syncope, trigeminal neuralgia, idiopathic orthostatic hypotension in 1 case. METHODS： Cerebrospinal fluid was collected from the involved patients using the lumbar puncture method. Proteins in the cerebrospinal fluid were separated by two-dimensional electrophoresis. MAIN OUTCOME MEASURES： Characteristics of protein electrophoresis patterns were analyzed, differentially expressed proteins were detected using matrix-assisted laser desorption ionization time of flight mass spectrometry, and protein data were analyzed in the Mascot database. RESULTS： Five protein electropherograms were analyzed by PDQuest 8.0, and （789 ± 32） protein spots were observed. There were significant differences in four protein spots in each of the PD sub-groups compared with the non-disease and control groups. Expression was down-regulated in three protein spots and up-regulated in one protein spot; 100% repetition rate was observed in four protein spots. According to the Mascot database, protein spots with down-regulated expression were as follows： DNA-guided RNA polymerase III subunit RPC5 （score： 50 points）; double serine, threonine, and tyrosine protein kinase （score： 64 points, P 〈 0.05）; activity-regulated cytoskeleton-associated protein （score： 58 points, P 〈 0.05）. However, G2 mitotic-specific cyclin was up-regulated （score： 84 points, P 〈 0.05）. CONCLUSION： Differential protein expression in the cerebrospinal fluid of PD patients was detected by two-dimensional electrophoresis-mass spectrometry, revealing changes in DNA-guided RNA polymerase III subunit RPC5, double serine, threonine, and tyrosine protein kinase, activity-regulated cytoskeleton-associated protein, and G2 mitotic cell cyclin, with good reproducibility.

Protein expression of sensory and motor nerves Two-dimensional gel electrophoresis and mass spectrometry

The present study utilized samples from bilateral motor branches of the femoral nerve, as well as saphenous nerves, ventral roots, and dorsal roots of the spinal cord, to detect differential protein expression using two-dimensional gel electrophoresis and nano ultra-high performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry techniques. A mass spectrum was identified using the Mascot search. Results revealed differential expression of 11 proteins, including transgelin, Ig kappa chain precursor, plasma glutathione peroxidase precursor, an unnamed protein product （gil55628）, gfyceraldehyde-3-phosphate dehydrogenase-like protein, lactoylgfutathione lyase, adenyfate kinase isozyme 1, two unnamed proteins products （gil55628 and gi11334163）, and poly（rC）-binding protein 1 in motor and sensory nerves. Results suggested that these proteins played roles in specific nerve regeneration following peripheral nerve injury and served as specific markers for motor and sensory nerves.

Proteome analysis of round-headed and normal spermatozoa by 2-D fluorescence difference gel electrophoresis and mass spectrometry

Globozoospermia is a severe form of teratozoospermia characterized by round-headed spermatozoa with an absent acrosome, an aberrant nuclear membrane and midpiece defects. Globozoospermia is diagnosed by the presence of 100% round-headed spermatozoa on semen analysis, and patients with this condition are absolutely infertile. The objective of this study was to investigate the differences in protein expression between human round- headed and normal spermatozoa. Two-dimensional （2-D） fluorescence difference gel electrophoresis （DIGE） coupled with mass spectrometry （MS） was used in this study. Over 61 protein spots were analysed in each paired normal/round-headed comparison, using DIGE technology along with an internal standard. In total, 35 protein spots identified by tandem mass spectrometry （MS/MS） exhibited significant changes （paired t-test, P 〈 0.05） in the expression level between normal and round-headed spermatozoa. A total of nine proteins were found to be upregulated and 26 proteins were found to be downregulated in round-headed spermatozoa compared with normal spermatozoa. The differentially expressed proteins that we identified may have important roles in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism and spermatozoa motility.

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.

Method Development of Enantiomer Separations by Affinity Capillary Electrophoresis,Cyclodextrin Electrokinetic Chromatography and Capillary Electrophoresis-Mass Spectrometry

Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection sensitivity was observed under high concentration of the protein in running solutions. The partial filling technique was practically useful to solve the serious problem. It allowed operation at high protein concentrations, such as 500 μmol/L, without the detection problem. Charged CDs had several advantages for the enantiomer separations over neutral ones. Strong electrostatic interactions between a charged CD and oppositely charged analytes should be effective for the formation of the complex. A large difference in electrophoretic mobility between the free analyte and the inclusion complex should also enhance the enantiomeric resolution. In CE mass spectrometry (CE MS), the partial filling technique was applied to avoid the introduction of nonvolatile chiral selectors into the CE MS interface. By replacing the nonvolatile electrolytes in the running buffer by volatile ones, the separation conditions employed in CE with the UV detection method could be transferred to CE MS.

Two-dimensional Electrophoresis Analysis of Proteins in Response to Cold Stress in Extremely Cold-resistant Winter Wheat Dongnongdongmai 1 Tillering Nodes

The overwintering survival ratio of the cultivar Dongnongdongmai 1 with strong cold-resistance in paramos of Heilongjiang Province in China are over 85%. The tillering nodes are the most important organs for overwintering survival of winter wheat, because there are more substances associated with cold resistance in tillering nodes than those in leaves and roots. Proteins in the tillering nodes of the cold-resistant cultivar Dongnongdongmai 1 grown under field conditions with or without any lowtemperature stress were analyzed by 2-dimensional electrophoresis and identified by mass spectrometry. In the range of pH 4-7, the expression of 37 proteins showed obvious difference （±more than two fold） in the proteomic maps of cold-stressed and non-stressed tillering nodes, including a new protein spot. All proteins exhibiting the difference in expression were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, followed by a database search for protein identification and function prediction. Five groups of proteins were confirmed, namely stress-related proteins （22%）, metabolism-associated proteins （35%）, and signaling molecules （24%）, cell wall-binding proteins （5%）, unclear proteins （14%）. This indicated that tillering node cells supported the energy requirements of plant growth and stress resistance by signal transduction adapting to metabolism and structure.

Establishment and Preliminary Application of Two dimensional Electrophoresis and Mass Spectrometry in Ovary Study

Objective To apply two-dimensional electrophoresis and mass spectrometry in the ovary proteome researchMethods Protein extractions from mouse ovaries were run in IPGphor isoelectric focus system with 11 cm and 24 cm IPG strips respectively (pH 3～10, 0.3 mm thick), then the protein spots were identified by mass spectrometry.Results The ovary protein exactions separated by two-dimensional electrophoresis have got high resolution, and identifing protein by mass spectrometry was highly efficient and facilitly. These two techniques should facilitate further investigation of female reproduction proteome research.Conclusion These two rapid high resolutions and efficient techniques have a variety of applications foreground in female reproduction proteome pattern research.

Analyzing crude oils from the Junggar Basin(NW China) using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry(GC×GC-TOFMS)

As a new technology of analyzing crude oils,comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry(GC×GCTOFMS) has received much research attention.Here we present a case study in the Junggar Basin of NW China.Results show that the hydrocarbons,including saturates and aromatics,were all well-separated without large coelution,which cannot be realized by conventional one-dimensional GC-MS.The GC×GC technique is especially effective for analyzing aromatics and low-to-middlemolecular-weight hydrocarbons,such as diamondoids.The geochemical characteristics of crude oils in the study area were investigated through geochemical parameters extracted by GC×GC-TOFMS,improving upon the understanding obtained by GC-MS.Thus,the work here represents a new successful application of GC×GCTOFMS,showing its broad usefulness in petroleum geochemistry.

Specific protein expression in a rat model of early focal cerebral ischemia:Fluorescent two-dimensional difference gel electrophoresis

BACKGROUND： The use of fluorescent two-dimensional difference gel electrophoresis （2D-DIGE） has been shown to compensate for the shortcomings of conventional two-dimensional gel electrophoresis, such as poor repeatability and large systematic errors. However, little information is presently available regarding the use of 2D-DIGE to investigate mechanisms of ischemic cerebrovascular diseases. Plasma and body fluids have been utilized in proteomic technology to study ischemic cerebrovascular diseases. OBJECTIVE： To perform proteomic analysis of fresh rat brain tissue in peripheral ischemic regions using 2D-DIGE 6 hours after middle cerebral artery occlusion （MCAO）, and to identify specific proteins closely associated with early ischemic cerebrovascular diseases. DESIGN, TIME AND SETTING： Proteomics-based, randomized, controlled, animal experiment was performed at the Laboratories of Neurology and Proteomics, Jilin University between January and April 2006. MATERIALS： 2, 3, 5-triphenyl tetrazolium chloride was purchased from Sigma, USA. Ettan DALTSix system, DeCyder DIA V5.0 differential analysis software, and Ettan matrix-assisted laser desorption/ionization time-of-flight mass spectrometer （MALDI-TOF-MS） were purchased from Amersham Bioscience, Sweden. METHODS： Eight healthy, male, Wistar rats were randomized to experimental and control groups, with four rats in each group. In the experimental group, rat models of focal cerebral ischemia were established by MCAO. In the control group, the internal and external carotid arteries were exposed and then immediately sutured, and the remaining procedures were identical to the experimental group. MAIN OUTCOME MEASURES： At 6 hours after cerebral ischemia, protein expression in the peripheral ischemia region of the experimental group was compared with the control group using 2D-DIGE. Protein spots that exhibited statistical differences between experimental and control groups with 〉 1.4 attributable risk were screened using DeCyder DIA V5.0 differential analysis software. Differential proteins were identified using MALDI-TOF-MS. RESULTS： Triphenyl tetrazolium chloride staining results revealed pink, normal brain tissue and white, ischemic brain tissue, suggesting successful MCAO establishment. The average matching rate of four 2D-DIGE gels was 92.4%. There were （1 758 ± 43） protein spots on each gel, with similar distribution modes. At 6 hours after focal cerebral ischemia, 13 protein spots exhibited marked expression changes, including significantly increased （n = 7） and decreased （n = 6） expression （P 〈 0.05）. MALDI-TOF-MS results revealed two differential protein spots： a-tubulin and heat shock protein 27, which were significantly decreased in the experimental group compared with the control group （P 〈 0.05）. CONCLUSION： Thirteen protein spots with expression changes were revealed by 2D-DIGE proteomics technology. Of them, a-tubulin and heat shock protein 27 expressions were markedly decreased during the early stage of cerebral ischemia. These two proteins were presumed to be proteins associated with early ischemic cerebrovascular diseases.

Resolving the geometric structure of trastuzumab by mobility capillary electrophoresis and native mass spectrometry

Available online Immunoglobulins G(IgGs)are Y-shaped globular proteins,however,their high flexibility and heterogeneity pose great challenges to their structure and conformation determinations.Geometric structure of IgG closely correlates to its biofunctions,such as the antibody escape of human immunodeficiency virus(HIV)could attribute to the distance mismatch between the ends of two Fab arms(antigen-binding sites)and envelope glycoprotein spikes on virion surface.Herein,we report the first use of mobility capillary electrophoresis(MCE)and native mass spectrometry(nMS)to resolve the internal geometric structure and conformation of an IgG(trastuzumab)in solution phase.After proteolysis,the ellipsoid dimensions of IgG and its subunits were measured by MCE-nMS experiments.IgG was then reconstructed,in which the sizes and relative positions of these three subunits in three-dimensional space were characterized.It was found that the two Fab arms have an angle of~102.1°and a distance of~11.0 nm between the two antigen-binding sites under native condition,and the Fc arm was tilted~16.0°towards one of the Fab arms.Fc was not on the plane of Fab-Fab,but has an angle of no larger than 103.1°.Under acidic environment(pH 3.0),each subunit of the IgG would unfold into larger dimensions,and the angles between these subunits also change.With great potential for tumor imaging and therapy,the structure of F(ab')_(2)fragments was also measured and validated by molecular dynamic simulation.It was found that the electrostatic force among these three subunits and steric hindrance stemming from Fc help maintaining the angle between two Fab arms.

Two-dimensional electrophoretogram of acute brain injury-associated proteins Comparison between injured and normal cerebral cortex

BACKGROUND： To this date, specific molecular markers for early diagnosis and prognosis monitoring of craniocerebral injury in clinical medicine do not exist. Therefore, differential detection of specific proteins might play an important role in diagnosis and treatment of this type of brain injury. OBJECTIVE： To compare differential cerebral cortical protein expression of craniocerebral injury patients and normal subjects through the use of proteomics. DESIGN： Contrast observation. SETTING： Department of Neurosurgery, Xiangya Hospital of Central South University. PARTICIPANTS： Ten patients （6 males and 4 females, 20 58 years old）, with severe craniocerebral injury, were selected at the Department of Neurosurgery, Xiangya Hospital of Central South University, from June 2004 to December 2006. All patients were diagnosed with CT test and Glasgow test （scores 〈 8）. Surgery was performed 4-12 hours after craniocerebral injury, and injured cortical tissues of the frontal and temporal lobes were resected for sampling. At the same time, control cortical tissues were collected from frontal and temporal lobes of 2 epileptic patients who underwent hippocampus-nucleus amygdala resection, and 2 lateral ventricular tumor patients who underwent tumor resection. The participants and their relatives provided confirmed consent, and this study received confirrned consent from the local ethics committee. METHODS： Ten samples from injured patients and 4 normal samples were compared through the use of proteomics. Total protein was separated by using two-dimensional electrophoresis with immobilized pH gradients, and the differential protein expressions were compared using image analysis after blue-sliver staining. Differential protein spot expressions were analyzed with a matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI/TOF MS） and electrospray ionization-quadrupole time of flight mass spectrometry （ESI-Qq TOF MS）. MAIN OUTCOME MEASURES：① Two-dimensional electrophoresis of protein from cerebral cortex; ② differential protein expression. RESULTS： ① Two-dimensional electrophoresis of protein from cerebral cortex： two-dimensional gel electrophoretogram, which is considered to have high resolution and consistent duplication, was performed on injured cortical tissues and normal cortical tissues. The image analysis system detected 21 differential protein spots. ② Differential protein spot expressions： mass spectrometry resulted in 17 differential protein spots that related to metabolic response, oxidative stress response, and signal transduction. CONCLUSION： MALDI/TOF MS and ESI-Qq TOF MS are exceptional methods for evaluating differential protein expression. Results from this study indicated 17 different craniocerebral injury-associated proteins.

A robust protein extraction method for two dimensional electrophoresis of silkworm proteins

Silkworm (Bombyx mori) is a species of agricultural importance, as well as a model organism for Lepidoptera insects. Proteomic method has been widely used in silkworm research, and a robust mass spectrometry-compatible protein extraction method is urgently needed. In this study, we adapted phenol extraction method to extract silkworm midgut protein, and coupled this method with pH 5 - 8 gel strip for two dimensional electrophoresis. The phenol extraction method significantly increased the resolution, as well as greatly reduced the background of two dimensional electrophoresis gels. In addition, this method was well compatible with mass spectrometry analysis. This is the first report that phenol extraction method is used for silkworm midgut protein extraction, and may be applied in other researches.

胆囊癌差异蛋白质组学分析

目的运用蛋白质组学经典技术,双向凝胶电泳、质谱鉴定及生物信息学分析在胆囊癌组、正常胆囊对照组中寻找差异蛋白,发现和鉴定胆囊癌相关的肿瘤标志物及潜在的治疗靶点。方法标本分两组:胆囊癌组-胆囊癌组标本59例;正常对照组-胆囊癌癌旁组织标本59例。组织样品通过洗涤、裂解、离心收集总蛋白样本,分装冻存。经过第一向等点聚焦、第二向SDS-PAGE电泳,凝胶经考马斯亮蓝染色显色后,用Gs-800光密度仪获得凝胶图像。经PD-Quest凝胶图像分析软件进行分析后,找出有差异的蛋白点。通过蛋白指纹谱鉴定(PMF),基质辅助激光解吸附质谱技术(MALDI-TOF)获得差异蛋白碎片的二级质谱,利用Biotools软件采集肽碎片质荷比信息;利用Mascot搜索引擎对数据库进行检索,鉴定差异蛋白质的氨基酸序列,获得相应特异蛋白质信息。结果发现66个差异表达蛋白。其中在胆囊癌中上调表达蛋白21个,下调表达蛋白45个。21个差异蛋白得分大于56,在胆囊癌中上调表达的10个,下调表达的11个。主要包括膜联蛋白、膜突蛋白、热休克蛋白、波形蛋白、视黄醛脱氢酶1、半乳凝素等。结论上述蛋白可能在肿瘤的发生发展机制中起着重要的作用,是诊断、治疗的潜在靶点,值得后续进一步研究。

无鞘流毛细管电泳-电喷雾串联质谱用于面粉中荧光增白剂的高灵敏检测

食品中残留的过量荧光增白剂(FWAs)对人体健康存在潜在威胁,因此,开发准确、高灵敏的FWAs检测方法在食品安全监测方面具有重要意义。本工作提出了利用无鞘流接口的毛细管电泳-电喷雾串联质谱(CE-ESI-MS/MS)的方法,以实现面粉样品中6种FWAs的高灵敏检测。实验采用超声辅助液相萃取法进行样品前处理,以减小复杂样品中基质效应的干扰。以氯仿-甲醇(3∶2,v/v)溶液作为萃取剂,在30℃下对样品中的FWAs进行萃取。萃取完成后,经离心、氮气吹干后用氯仿-甲醇(1∶4,v/v)复溶后检测。无鞘流CE-ESI-MS/MS方法采用正离子(ESI+)和多反应监测(MRM)模式,利用二级质谱对6种目标物同时进行定性定量分析,从而提高方法的检测通量和灵敏度。结果显示,本方法具有较宽的线性范围、良好的线性关系和较低的方法检出限(0.04~0.67 ng/g),在实际样品中3个水平下的加标回收率良好(86.2%~103.7%),日间和日内重复性(RSD)分别不大于11.5%和10.2%。上述研究表明,本方法适用于复杂基质中多种FWAs的准确、高灵敏检测,在面粉样品的质量评估和FWAs的污染监控方面具有潜在的应用价值。

Congophilic fibrils in the glomeruli with polyclonal immunoglobulin gamma staining-another cause for diagnostic overlap:A case report

BACKGROUND Glomerulopathy with fibrillary deposits is not uncommon in routine nephropathology practice,with amyloidosis and fibrillary glomerulonephritis being the two most frequently encountered entities.Renal amyloid heavy and light chain(AHL)is relatively uncommon and its biopsy diagnosis is usually limited to cases that show strong equivalent staining for a single immunoglobulin(Ig)heavy chain and a single light chain,further supported by mass spectrometry(MS)and serum studies for monoclonal protein.But polyclonal light chain staining can pose a challenge.CASE SUMMARY Herein we present a challenging case of renal AHL with polyclonal and polytypic Ig gamma(IgG)staining pattern by immunofluorescence.The patient is a 62-yearold Caucasian male who presented to an outside institution with a serum creatinine of up to 8.1 mg/dL and nephrotic range proteinuria.Despite the finding of a polyclonal and polytypic staining pattern on immunofluorescence,ultrastructural study of the renal biopsy demonstrated the presence of fibrils with a mean diameter of 10 nm.Congo red was positive while DNAJB9 was negative.MS suggested a diagnosis of amyloid AHL type with IgG and lambda,but kappa light chains were also present supporting the immunofluorescence staining results.Serum immunofixation studies demonstrated IgG lambda monoclonal spike.The patient was started on chemotherapy.The chronic renal injury however was quite advanced and he ended up needing dialysis shortly after.CONCLUSION Tissue diagnosis of AHL amyloid can be tricky.Thorough confirmation using other available diagnostic techniques is recommended in such cases.

Identification of petroleum aromatic fraction by comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry

Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry(GC×GC-TOFMS) is commercially available in the 1990s,with the characteristics of large peak capacity,high resolution,high sensitivity,etc.However,its application to the petroleum and geological analyses is just emerging in China and overseas.In this research,the analytical method for petroleum aromatic fraction using GC×GC-TOFMS is set up,via the choice of the column system and optimization of setting parameters,such as temperature programming,modulation time,hot pulse time,flow rate of carrier gas,data acquisition rate and data processing.The results indicate that different polar compounds of aromatic fraction distribute as bands on structured GC×GC chromatogram.Within each band,homologous compounds appear as a roof-tile structure based on the number of substituent residues.The aromatic compounds are identified and characterized according to the GC×GC chromatogram and mass spectra.According to the polarity and the number of rings,aromatic compounds are spatially present on one chromatogram,which directly reflects the distribution characteristics of complex compounds of aromatic hydrocarbons.In addition,quantitative analysis is favored as some overlapped peaks on traditional GC-MS chromatogram have been separated completely on GC×GC.Some heterocyclic atom aromatic compounds at trace level can be clearly identified using this method,for polarity differences from other interfered aromatic compounds.The development of this method and chromatogram recognition offer petroleum geologists a practical example for the application performance of GC×GC-TOFMS.

Qualitative Analysis of A Sulfur-fumigated Chinese Herbal Medicine by Comprehensive Two-Dimensional Gas Chromatography and High-Resolution Time of Flight MassSpectrometry Using Colorized Fuzzy Difference Data Processing

To investigate the chemical transformation of volatile compounds in sulfur-fumigated Radix Angelicae Sinensis. A comprehensive two-dimensional gas chromatography (GCxGC) and high-resolution time-of-flight mass spectrometry (HR-TOF/MS) with colorized fuzzy difference (CFD) method was used to investigate the effect of sulfur-fumigation on the volatile components from Radix Angelicae Sinensis. Twenty-five compounds that were found in sun-dried samples disappeared in sulfur-fumigated samples. Seventeen volatile components including two sulfur-containing compounds were newly generated for the first time in volatile oils of sulfur-fumigated Radix Angelicae Sinensis. The strategy can be successfully applied to rapidly and holistically discriminate sun-dried and sulfur-fumigated Radix Angelicae Sinensis. GCxGC-HR-TOF/MS based CFD is a powerful and feasible approach for the global quality evaluation of Radix Angelicae Sinensis as well as other herbal medicines.

Un raveling the acetals as ageing markers of Chinese Highland Qingke Baijiu using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry combined with metabolomics approach

Objectives:The ageing process has a significant impact on the aroma of Chinese Baijiu,which could strengthen the desirable flavor characteristics and reduce the undesirable ones.The aim of this study was to observe the in itiation of mean in gful cha nges in volatile fracti on and locate the ageing markers during ageing storage of Chinese Highland Qingke Baijiu.Materials and Methods:Samples of Chinese Qingke Baijiu were aged for 0,1,2,3,4,5,6,7,8,9,10,and 11 mon ths before an alysis.The samples were isolated by liquid-liquid extraction and then analyzed by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.The acquired data were processed by untargeted and targeted metabolomics approach to locate the ageing markers.Results:The untargeted metabolomics analysis(hierarchical clustering analysis,HCA)shows that the chemical composition of Qingke Baijiu presents a statistically sigrdficant deviation from the reference scenario after 5 mon ths.Subsequently,supervised statistics analysis(orthogo nal partial least squares discrimination analysis)was performed to locate the markers,which changed sigrdficantly during ageing.Fifteen markers were located,and seven of them were acetals.Notably,1,1-diethoxy-propane,1,1-diethoxy-butane,and 1,1-diethoxy-3-methyl-butane are important contributors to the flavor of Chinese Baijiu.The identified markers were applied for the untargeted metabolomics(HCA),and the results revealed that these markers could divide the Qingke Baijiu into two ageing stages,0-5 months and 6-11 months.Conclusion:The results suggest that it is a valuable tool for monitoring the changes of volatile compounds and locating the age markers in Chinese Baijiu.