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.

A novel sample preparation method of matrix-assisted laser desorption/ionization time of flight mass spectrometry for polystyrene

A novel sample preparation method of matrix-assisted laser desorption/ionization mass spectrometry for polystyrene was reported. Compared to the conventional dried-droplet method, the efficiency of ionization and signal intensity of mass spectra were improved. The mechanism was also analyzed.

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.

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.

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.

Mode of interaction of calcium oxalate crystal with human phosphate cytidylyltransferase 1: a novel inhibitor purified from human renal stone matrix

Nephrolithiasis is a common clinical disorder, and calcium oxalate (CaOx) is the principal crystalline component in approximately 75% of all renal stones. It is widely believed that proteins act as inhibitors of crystal growth and aggregation. Acidic amino acids present in these proteins play a significant role in the inhibition process. In this study, interaction of cal-cium oxalate with human phosphate cytidylyltrans-ferase 1(CCT), a novel calcium oxalate crystal growth inhibitor purified from human renal stone matrix has been elucidated in silico and involvement of acidic amino acids in the same. As only sequence of CCT is available, henceforth its 3-D structure was modeled via Homology modeling using Prime module of Schrodinger package. Molecular dynamic simulation of modeled protein with solvation was done by mac-romodel (Schrodinger). The quality of modeled pro-tein was validated by JCSG protein structure valida-tion (PROCHECK & ERRAT) server. To analyze the interaction of modeled protein CCT with calcium oxalate along with role played by acidic amino acids, ‘Docking simulation’ was done using MOE–Dock. Interaction between calcium oxalate and CCT was also studied by substituting acidic amino acid in the active sites of the protein with neutral and positively charged amino acids. The in silico analysis showed the bond formation between the acidic amino acids and calcium atom, which was further substantiated when substitution of these acidic amino acids with alanine, glycine, lysine, arginine and histidine com-pletely diminished the interaction with calcium ox-alate.

基质辅助激光解吸电离飞行时间质谱快速检测生菜中嗜麦芽窄食单胞菌

目的评价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)在即食果蔬中微生物风险监测的应用价值,确定生菜中的危害微生物及蛋白指纹图谱特征。方法对成都地区50份生菜样品进行微生物的检测,对分离的嗜麦芽窄食单胞菌进行生化鉴定和MALDI-TOF MS鉴定,采用维恩分析(包括小于0.85‰偏差)对生菜中分离的嗜麦芽窄食单胞菌指纹图谱进行拟合,采用系统聚类法对分离的嗜麦芽窄食单胞菌指纹图谱进行聚类分析。结果50份生菜样品中检出11株嗜麦芽窄食单胞菌,MALDI-TOF MS获得其蛋白图谱拟合出特征性标识峰指纹图16组,与数据库中超级谱图比对有9组不同标识峰,为生菜分离菌特征峰;蛋白指纹图谱聚类结果表明嗜麦芽窄食单胞指纹图谱有一定来源指向性,但仍需更多数据修正。结论嗜麦芽窄食单胞菌在生菜中检出率较高,可作为危害风险特征微生物继续研究,MALDI-TOF MS可获得更多农产品中致病微生物特征信息,作为农产品危害识别微生物快速鉴定及差异性分析应更深入的研究和扩大数据量,并建立农产品微生物数据库系统。

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。

建立一种适合本实验室快速检测产KPC酶肺炎克雷伯菌的方法

目的运用基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF MS)检测产肺炎克雷伯菌碳青霉烯酶(KPC)耐碳青霉烯类肺炎克雷伯菌(CRKP),筛选并验证其特征峰,建立一种适用于本实验室快速检测产KPC酶型CRKP的方法。方法收集临床耐碳青霉烯类肺炎克雷伯菌39株,运用PCR方法检测出其中23株产KPC酶,再采用MALDI-TOF MS技术检测KPC酶阳性CRKP菌株,得到质谱图后用Flex Analyst软件分析筛选出其中特征峰,并进行重复性实验和临床验证评估。结果综合筛选出产KPC酶CRKP最佳特征峰为m/z 6432,特征峰验证实验和重复性实验的特异性和敏感性分别为96.67%和100%。临床验证评估特异性和敏感性分别为92.31%和100%。结论利用MALDI-TOF MS技术可检出产KPC酶CRKP的最佳特征峰为m/z 6432,经实验证实该特征峰特异性和敏感性高,可为临床快速诊断和治疗产KPC酶CRKP提供理论基础。

核酸MALDI-TOF MS检测在肺结核诊断中的价值

目的探讨基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)在疑似肺结核(PTB)诊断中的应用价值及其阳性结果的影响因素,以期为临床治疗提供参考。方法选取甘肃省第二人民医院(西北民族大学附属医院)2022年8月—2023年3月收治的疑似PTB患者77例为研究对象。所有患者肺泡灌洗液(BALF)均接受MALDI-TOF MS检测和结核分枝杆菌(MTB)培养。根据MTB培养结果,对核酸MALDI-TOF MS检测进行诊断效能分析,并探讨核酸MALDI-TOF MS检测阳性的影响因素。结果核酸MALDI-TOF MS检测结果显示,MTB阳性共37例(48.1%,37/77),40例阴性(51.9%,40/77)。以培养结果为金标准,核酸MALDI-TOF MS的灵敏度、特异度、阳性预测值(PPV)、阴性预测值(NPV)和准确度分别为90.9%、84.1%、81.1%、92.5%和87.0%,采用Kappa检验法评价核酸MALDI-TOF MS检测和MTB培养法的一致性,结果显示Kappa值为0.739(P<0.05)。2组患者在是否有糖尿病、身体质量指数(BMI)及降钙素原(PCT)方面比较,差异均有统计学意义(P<0.05)。多因素Logistic回归分析结果显示:有糖尿病和PCT异常为核酸MALDITOF MS阳性的独立危险因素(P<0.05)。结论核酸MALDI-TOF MS检测对PTB的早期诊断具有重要价值,可为患者的规范用药提供更好的指导,即为患者的治疗制定更为精准、有效的治疗方案,从而提高治疗效果、改善患者生活质量。

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。

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。

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。

人面部皮肤来源疑难鉴定细菌的分子鉴定方法探索

目的比较分析全自动生化鉴定系统、MALDI-TOF MS、16S rDNA测序和WGS技术对皮肤来源的24株细菌的鉴定结果,为疑难菌种的准确鉴定提供方法参考。方法针对24株人面部皮肤来源的疑难鉴定菌株,分别采用VITEK 2 Compact生化鉴定、MALDI-TOF MS、16S rDNA系统发育分析以及基于WGS的ANI分析进行菌种鉴定,并评估各鉴定方法的准确度。结果MALDI-TOF MS方法在细菌种水平的鉴定准确度为8.3%,在属水平的鉴定准确度为70.8%;16S rDNA序列分析方法在属水平的鉴定结果与WGS一致,但在种水平的鉴定结果仍存在较大差异。以目的菌株与数据库推荐参考菌种的ANI值为评价标准,仅12株菌(50.0%)能够准确鉴定到种水平。结论针对皮肤来源的疑难鉴定细菌,以VITEK 2 Compact为代表的生化鉴定方法和MALDI-TOF MS的鉴定准确度有限,综合使用16S rDNA测序和WGS等分子鉴定技术能提高鉴定准确度。

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。