Analysis of Single Nucleotide Polymorphism in Human Paraoxonase 1 Gene(Q192R) with Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Introduction Single nucleotide polymorphisms （SNPs） are the most abundant DNA markers in the human genome occurring at a frequency of one in every 500--1000 nucleotides. A variety of methods have been used for the analysis of single nucleotide polymorphisms, including restriction fragment length polymorphism （RFLP）, direct sequencing by using laser-induced fluorescence detectionTM, fluorescence energy transfer, MALDI-TOF MS combined with primer extension or invasive cleavage, and fluorescence polarization. During the past two decades, mass spectrometry has become a very popular tool in the analysis of biomolecules and is perfectly suited to the analysis of single nucleotide polymorphisms （SNPs） due to its speed, low cost, and accuracy. In this work, we used MALDI TOF mass spectrometry to detect the fragments of restriction endonuclease hydrolysis of PCR products flanking a SNP located at paraoxonase 1（Q192R）. Compared with electrophoresis, this method requires less time of analysis and possess a higher accuracy.

MicrobMatcher: a microbial comparison software based on matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry

Matrix-assisted Laser Desorption/Ionization with Time-of-flight Mass Spectrometry (MALDI-TOFMS) was investigated as a method for the rapid identifica-tion of species. Current demand in microbial identi-fication is how to compare unknown strains to the known one quickly, semi-automatically and accurately. In this paper, we present a software tool that allows flexibly microbial matching in a user-friendly way, by letting the users to customize comparison parameters including: in vitro transcription enzyme, mass tolerance,minimum fragment length, intensity threshold and corresponding weights. We provide three spectral scoring functions to compute the affin-ity between the species. Therefore, the precision of microbial comparison increases. To test and verify this tool, we employed experimental spectral data based on MALDI-TOFMS and the gene sequences of E.coli and Salmonella. This software is written in Java for cross-platform intention.

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.

Detection of lung adenocarcinoma using magnetic beads based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry serum protein profiling

Background Recently, due to the rapid development of proteomic techniques, great advance has been made in many scientific fields. We aimed to use magnetic beads （liquid chip） based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS） technology to screen distinctive biomarkers for lung adenocarcinoma （adCA）, and to establish the diagnostic protein profiles. Methods Using weak cation exchange magnetic beads （MB-WCX） to isolate and purify low molecular weight proteins from sera of 35 lung adCA, 46 benign lung diseases （BLDs） and 44 healthy individuals. The resulting spectra gained by anchor chip-MALDI-TOF-MS were analyzed by ClinProTools and a pattern recognition genetic algorithm （GA）. Results In the working mass range of 800-10 000 Da, 99 distinctive peaks were resolved in lung adCA versus BLDs, while 101 peaks were resolved in lung adCA versus healthy persons. The profile gained by GA that could distinguish adCA from BLDs was comprised of 4053.88, 4209.57 and 3883.33 Da with sensitivity of 80%, specificity of 93%, while that could separate adCA from healthy control was comprised of 2951.83 Da and 4209.73 Da with sensitivity of 94%, specificity of 95%. The sensitivity provided by carcinoembryonic antigen （CEA） in this experiment was significantly lower than our discriminatory profiles （P 〈0.005）. We further identified a eukaryotic peptide chain release factor GTP-binding subunit （eRF3b） （4209 Da） and a complement C3f （1865 Da） that may serve as candidate biomarkers for lung adCA. Conclusion Magnetic beads based MALDI-TOF-MS technology can rapidly and effectively screen distinctive proteins/polypeptides from sera of lung adCA patients and controls, which has potential value for establishing a new diagnostic method for lung adCA.

Studies on extracting solutions of endohedral rare-earth metallofullerenes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Thirteen extracting solutions of rare-earth metallofullerenes containing La,Ce,Pr,Nd Sm,Eu,Gd,Tb,Dy,Ho,Er,Tm and Yb respectively have been investigated by means of matrix-assisted laser desorpuon/ ionization time-of-flight mass spectrometry.The influences of the positive-ion/negative-ion mode,laser intensity,ma trix and mass discrimination to the analytical results are studied,based on which the optimal analytical conditions have been determined.The results show that the extracting solutions contain large quantities of rare-earth metallofullerenes besides empty fullerenes.On the basis of comparing their relative intensities,the different structure stabilities and solubilities of metallofullerenes with different rare-earth metals encapsulated into the fullerene cages,as well as some possible reasons to those differences,are discussed.

Detection of Sialylated N-Linked Glycans by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Native and methyl-esterified sialylated glycans were analyzed with 2,4,6-trihydroxyacetophenone（THAP）and 2,5-dihydroxybenzoic acid（DHB）as matrix by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer（MALDI-TOF MS）.High quality negative-ion spectra of commercial sialylated glycan were obtained with THAP as matrix.Detection limit of the glycan was less than 0.1 pmol.After methyl esterification of sialic acid（SA）residue,sialylated glycans were detected sensitively in the positive-ion mode using DHB as matrix.Neutral and sialylated glycans from the mixture of asialofetuin and fetuin were methylesterified and simultaneously recognized in one manipulation.Methyl esterification of SA residue offers a convenient and sensitive way to identify the structure of N-linked glycans for glycan profiling.

Mass spectrometry-based serum peptide profiling in hepatocellular carcinoma with bone metastasis

AIM:To investigate the potential of serum peptides as a diagnostic tool for hepatocellular carcinoma(HCC)with bone metastasis.METHODS:Matrix-assisted laser desorption ionization-time of flight mass spectrometry(MALDI-TOF-MS)was used to characterize the serum peptide profile of HCC patients with bone metastasis.Serum samples from 138 HCC patients(66 cases with and 72 cases without bone metastasis)were randomly assigned into a training set(n=76)and a test set(n=62).Differential serum peptides were examined using ClinProt magnetic bead-based purification followed by MALDITOF-MS.The sequences of differentially expressed serum peptides were identified using liquid chromatography-mass spectrometry.A diagnostic model was established using a learning algorithm of radial basis function neural network verified by a single blind trial.Receiver operating characteristic(ROC)analysis was performed to evaluate the diagnostic power of the established model.RESULTS:Ten peptide peaks were significantly different between HCC patients with or without bone metastasis(P<0.001).Sequences of seven peptides with mass to charge ratios(m/z)of 1780.7,1866.5,2131.6,2880.4,1532.4,2489.8,and 2234.3 were successfully identified.These seven peptides were derived from alpha-fetoprotein,prothrombin,serglycin,isoform2 of inter-alpha-trypsin inhibitor heavy chain H4,isoform 1 of autophagy-related protein 16-2,and transthyretin and fibrinogen beta chains,respectively.The recognition rate and predictive power of a diagnostic model established on the basis of six significant peptides(m/z for these six peptides were 1535.4,1780.7,1866.5,2131.6,2880.4,and 2901.9)were 89.47%and82.89%,respectively.The sensitivity and specificity of this model based upon a single blind trial were 85.29%and 85.71%,respectively.ROC analysis found that the AUC(area under the ROC curve)value was 0.911.CONCLUSION:Our study suggested that serum peptides may serve as a diagnosis tool for HCC bone metastasis.

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.

Identification of serum biomarkers for diagnosing stage Ⅰ lung adenocarcinoma by MALDI-TOF mass spectrometry

Objective To identify specific biomarkers that could improve early diagnosis of lung adenocarcinoma using matrix-assisted laser desorption/ionization (MALDI) technology. Methods Serum samples were isolated from 17 patients with stage Ⅰ lung adenocarcinoma and 17 age-and sex-matched healthy controls,and the serum proteomic profiles were obtained by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Results Compared with healthy control group,two highly expressed potential biomarkers were identified with the relative molecular weights of 6 631.64 Da and 4 964.21 Da. The two best novel protein peaks were automatically chosen for the system training and the development of the constructed model. The constructed model was then used to test an independent set of masked serum samples from 15 lung adenocarcinoma patients and 22 healthy individuals. The analysis yielded a sensitivity of 93.3%,and a specificity of 95.5%. Conclusion These results suggest that MALDI-TOF-MS ProteinChip technology is a quick,convenient,and high-output analyzing method that is capable of selecting several relatively potential biomarkers from the serum of lung adenocarcinoma patients and may have a clinical value in the future,and will provide clues to identifying new serologic biomarkers of lung adenocarcinoma.

磁性氮化碳复合材料的制备及其对磷酸化肽的富集

蛋白质的磷酸化在细胞信号传导和疾病发生发展中起着重要作用,但磷酸化的动态变化和低丰度的特点使得对其直接分析有着较大的困难。为了解决磷酸化肽难离子化、检测丰度低的瓶颈问题,本研究制备了一种磁性氮化碳复合材料,结合基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS),建立了一种对复杂样品中低丰度磷酸化肽富集与分析的方法。采用电子显微镜、红外光谱分析及X射线衍射分析等手段对合成的磁性氮化碳材料进行表征。以酪蛋白酶解产物为实验模型,发现磁性氮化碳材料能够实现对磷酸化肽的高选择性富集和高灵敏度检测,检出限为0.1 fmol。选择脱脂牛奶、人唾液和人血清为实际分析样品,发现磁性氮化碳材料对微量蛋白生物样品中磷酸化肽的分析具有较高的应用潜力。

饮用水中高分子量消毒副产物的检测识别方法

消毒是饮用水处理过程中的重要步骤,但普遍采用的氯系消毒剂在杀灭细菌病毒的同时,能产生大量具有“三致”效应的卤代消毒副产物(DBPs),严重威胁了饮用水的化学安全.研究人员已在饮用水中陆续检测并识别出700多种DBPs,但这些已知DBPs均为分子量小于800 Da的低分子量DBPs,目前对高分子量DBPs的认识还较为有限.本研究建立了基于基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)的高分子量DBPs检测方法,发现在基质为2,5-二羟基苯甲酸,阳离子化试剂为三氟乙酸钠,使用三明治法点靶,反射-正离子模式,90%激光强度时,信噪比和信号重现性达到最优,信噪比之和达到了136.2,变异系数(CV)则为4.77%.利用上述方法,在模拟饮用水中检测到5种新的高分子量DBPs.在此基础上,通过同位素模式分析、TOF/TOF串联质谱和数据库验证,建立了针对未知高分子量DBPs的分子式/结构式识别方法,确定新的高分子量DBPs为寡糖羧酸类物质.

血培养阳性样本快速菌种鉴定和药物敏感性分析

目的分析分离胶离心富集和5 h培养2种前处理方法在血培养阳性样本快速菌种鉴定和快速药物敏感性分析中的临床应用价值。方法收集2021年10月—2022年10月宁波大学附属第一医院报阳的血培养样本211例,分别采用分离胶离心富集和5 h培养(快速方法),以及12 h培养(标准方法)3种方法进行前处理。采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS,简称MS)对3种方法处理的血培养阳性样本进行快速菌种鉴定。采用自动化鉴定药敏仪对156例需氧瓶报阳血培养样本进行药物敏感性试验。以常规培养(18~24 h)+MS鉴定结果为标准,比较5 h培养+MS和分离胶离心富集+MS 2种快速方法菌株鉴定准确率。以培养24 h后仪器法药物敏感性试验结果为标准,判断5 h培养和分离胶离心富集后仪器法药物敏感性试验结果与标准方法的一致率。结果2种快速方法鉴定准确率最高的菌种为粪肠球菌,分别为100.00%、87.50%;5 h培养+MS对链球菌属的鉴定准确率较低(33.33%)。2种快速方法对肠杆菌科和非发酵菌的药物敏感性分析结果与标准培养+MS的药物敏感性分析结果一致性分别超过73.00%和85.00%;对葡萄球菌属和肠球菌属部分抗菌药物的药物敏感性分析结果一致性达100.00%;对万古霉素的药物敏感性分析结果一致性最低,为70.73%。结论5 h培养+MS和分离胶离心富集+MS可快速判断血培养阳性样本病原菌种类及其耐药性,准确率较高,适合在临床微生物实验室推广。

MALDI-TOF MS直接靶板微滴生长测定法在CRKP快速检测中的应用

目的探讨基于基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)的直接靶板微滴生长测定(DOT-MGA)法快速筛查碳青霉烯类耐药肺炎克雷伯菌(CRKP)的准确性和临床应用价值。方法收集2020—2021年连云港市第一人民医院肺炎克雷伯菌临床分离株251株,其中美罗培南敏感123株,CRKP128株。采用DOT-MGA法对4和6μL 2种取样量的所有菌株进行美罗培南耐药性检测。采用微量肉汤稀释法测定菌株的美罗培南最小抑菌浓度(MIC)。以微量肉汤稀释法耐药性分析结果为标准,评价DOT-MGA法耐药性检测结果的准确性。结果以微量肉汤稀释法为标准,孵育生长4 h后,DOT-MGA法2种取样量的敏感性和阴性预测值均为100.0%,其中4μL取样量检测CRKP的特异性为92.5%,阳性预测值为92.8%;6μL取样量检测CRKP的特异性为89.8%,阳性预测值为90.1%。123株美罗培南敏感株的美罗培南MIC_(50)为0.125μg·mL^(-1),MIC_(90)为0.25μg·mL^(-1);128株美罗培南耐药株的MIC_(50)和MIC_(90)均为≥128μg·mL^(-1)。DOT-MGA法与微量肉汤稀释法的一致性分析结果显示,4μL加样量Kappa值为0.88,6μL加样量Kappa值为0.83。结论基于MALDITOF MS的DOT-MGA法筛查CRKP具有较高的准确性,且简便、快捷,可用于快速检测CRKP。

基质辅助激光解吸电离-飞行时间质谱法快速测定米饭和乳制品中蜡样芽孢杆菌呕吐毒素Cereulide

建立基质辅助激光解吸电离-飞行时间质谱(matrix-assisted laser desorption ionization time-of-flight mass spectrometry,MALDI-TOF MS)法分析蜡样芽孢杆菌呕吐毒素Cereulide的快速检测方法。分析了Cereulide标准物质的裂解规律,考察基质种类、点靶方式、基质溶剂和用量、激光强度等对Cereulide的质谱信号强度的影响,并进行方法学验证和实际样品检测。结果表明,选择以α-氰基-4-羟基肉桂酸为基质,均匀分散在50%乙腈-水(含0.1%三氟乙酸)中,采用先点基质再点样品的点样方式,反射线性正离子模式下选择激光强度70%进行食品中MALDI-TOF MS筛查时,可检测到Cereulide的[M+Na]^(+)和[M+K]^(+)目标离子峰,此质谱信号稳定、强度高、响应重复性好。方法学验证结果表明,Cereulide的[M+Na]^(+)和[M+K]^(+)目标离子峰在5~100 ng/mL范围内线性好,相关系数(r)>0.99;方法的检出限分别为3.0 ng/g和5.0 ng/g;对米饭和牛乳样品的加标回收率为73.3%~118.2%,相对标准偏差为0.3%~10.9%(n=6)。本方法分析速度快、准确性高、灵敏度好、抗干扰能力强、无需内标即可实现对米饭和乳制品中Cereulide的批量筛查和检测。

半夏炮制过程中毒性凝集素蛋白的基质辅助激光解吸电离飞行时间质谱分析

基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)受灵敏度和重复性的影响,对完整蛋白的定量分析存在困难。本研究采用纤维素纳米晶体(CNC)辅助MALDI-TOF MS,结合内标法定量分析半夏炮制过程中半夏凝集素(PTL),通过优化CNC辅助样本制备的时长和比例,以麦胚凝集素(TVL)为内标,直接分析不同炮制程度半夏各炮制品及其浸泡上清液中PTL含量。结果表明,CNC辅助MALDI样本制备有效提升了MALDI-TOF MS定量分析的线性和重复性。随着炮制时间的延长,姜半夏、清半夏和法半夏炮制品的PTL含量逐渐下降,浸泡上清液中PTL含量逐渐上升。其中,由于pH值的改变,法半夏炮制品浸泡上清液中PTL含量呈先上升后下降的趋势。此外,将该方法应用于不同饮片生产企业的多批次半夏炮制品饮片中,均未检出PTL。该方法操作简便,可有效分析半夏炮制过程中PTL含量的变化,为研究半夏炮制过程对PTL的影响提供了参考。

MALDI-TOF MS法快速鉴定散装即食食品中3种食源性致病菌

本研究旨在利用基质辅助激光解吸电离飞行时间质谱法(Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry,MALDI-TOF MS)快速检测并鉴定散装即食食品中的沙门氏菌、金黄色葡萄球菌和蜡样芽孢杆菌3种食源性致病菌。通过对点样方式、前处理方式、不同培养基及培养时间等各项实验条件的优化筛选确定最佳方法,并考察其检测限和稳定性,同时用所建立的方法与多重PCR法、VITEK 2等生化鉴定法进行结果比对,比较三者鉴定结果的一致性,最后进行批量食品样本检测来探寻MALDI-TOF MS鉴定法在散装即食食品中的适用性。结果表明,三种目标菌同一批次及不同传代次数的鉴定得分均大于95%,变异系数均小于2%,所建立的方法重复性和稳定性良好,且检测限为10^(5)CFU/mL,MALDI-TOF MS菌株鉴定结果与多重PCR法、生化鉴定法结果高度一致,且比二者检测用时更短,更加特异高效;在点样方式上,相较于夹心法与混合干滴法,三种覆盖法点样平均鉴定得分更高,均为99.9%,其中1μL+1μL覆盖法点样可获得的平均峰数目更多,特征峰更复杂且强度更高;而在前处理方式上,甲酸乙腈法与其他两种菌体处理方法相比可获得更多的出峰量和更高的峰强度,平均鉴定得分均在98%以上,且与数据库中图谱匹配度更高。综上所述,MALDI-TOF MS法在致病菌鉴定方面不仅灵敏特异、准确快速,且操作简便、结果直观,作为国标法的有力补充,为食源性致病菌的分析鉴定与分型溯源提供了新思路。

基质辅助激光解吸电离飞行时间质谱在食源性致病菌鉴定中的应用

基质辅助激光解吸电离飞行时间质谱(matrix assisted laser desorption ionization time of flight mass spectrometry,MALDI-TOF MS)作为近年来发展的新型食源性致病菌鉴定技术,具有灵敏、准确、检测速度快等优点,该技术为食品病原微生物靶标性监测和食品安全事件应急检验提供了一种高效的鉴别技术参考,在保障民众生命健康和经济社会发展上发挥了重要作用。本文检索了近年来国内外MALDI-TOF MS技术在食源性致病菌检测中的相关研究案例,简要综述了MALDI-TOF MS检测原理、工作流程,影响鉴定结果的主要因素,介绍了MALDI-TOF MS技术应用于食源性致病菌检测中的实际案例,在此基础上分别从参考菌株数据库建设、标准化程序规范等方面对MALDI-TOF MS技术在食源性致病菌检测领域的未来研究方向进行展望,以期为后续食品安全检测及快速监管食源性致病菌污染提供技术支持。

MALDI-TOF MS技术在临床微生物检验中的应用

随着科学技术的发展,新技术在医学检验中的应用越来越广泛。相较于传统微生物鉴定繁琐、耗时的工作流程,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS,简称MS)技术因其快速、灵敏、简便、省时和特异性高等特点,在菌种鉴定、耐药监测、突变菌株鉴别等方面发挥巨大的作用,逐渐成为临床微生物实验室不可或缺的微生物鉴定工具。虽然MS技术鉴定菌种高度依赖数据库,也在一定程度上受限于临床样本质量,但其在临床微生物检验中仍有较大的空间待开发。文章对MS技术在临床微生物检验中的应用进行综述。

宁夏桶装水中铜绿假单胞菌的污染情况、耐药性及同源性分析

为综合评价宁夏桶装水生产企业铜绿假单胞菌污染情况、耐药性及系统进化关系,确定污染来源,提升产品质量,该研究对企业各生产环节的水样进行铜绿假单胞菌检测,并采用全自动微生物鉴定系统(VITEK-2)、基质辅助激光解吸电离-飞行时间质谱仪(Matrix Assisted Laser Desorption Ionization-time of Flight Mass Spectrometry,MALDI-TOF MS)、16S rRNA测序法对分离菌株进行鉴定。铜绿假单胞菌的耐药性实验应用微生物全自动鉴定及药敏系统检测,采用16S rRNA基因序列测序构建系统发育树,MALDI-TOF MS进行同源性分析。结果表明,VITEK-2、MALDI-TOF MS、16S rRNA鉴定结果与常规方法分离的24株阳性菌株鉴定结果100%相符。耐药性结果表明,24株铜绿假单胞菌中2株为耐药菌株,分别是5-B-1,7-C-5,耐药率为8.3%,说明水源性菌株的耐药水平总体偏低。质谱结果表明来自同一地区的阳性菌株亲缘关系较近;16S rRNA系统发育表明铜绿假单胞菌分离株与地域不存在明显的相关性,但同一来源的铜绿假单胞菌属于同一簇,其来源主要为水源。该研究为桶装水中铜绿假单胞菌的污染溯源积累了数据,为进一步研究菌种污染溯源提供理论依据。