The isolation and identification of apolipoprotein C-I in hormone-refractory prostate cancer using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry

Androgens play a central role in prostate cancer pathogenesis, and hence most of the patients respond to androgen deprivation therapies. However, patients tend to relapse with aggressive prostate cancer, which has been termed as hormone refractory. To identify the proteins that mediate progression to the hormone-refractory state, we used protein-chip technology for mass profiling of patients＇ sera. This study included 16 patients with metastatic hormone-refractory prostate cancer who were initially treated with androgen deprivation therapy. Serum samples were collected from each patient at five time points： point A, pre-treatment; point B, at the nadir of the prostate- specific antigen （PSA） level; point C, PSA failure; point D, the early hormone-refractory phase; and point E, the late hormone-refractory phase. Using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, we performed protein mass profiling of the patients＇ sera and identified a 6 640-Da peak that increased with disease progression. Target proteins were partially purified, and by amino acid sequencing the peak was identified as a fragment of apolipoprotein C-I （ApoC-I）. Serum ApoC-I protein levels increased with disease progression. On immunohistochemical analysis, the ApoC-i protein was found localized to the cytoplasm of the hormone-refractory cancer cells. In this study, we showed an increase in serum ApoC-I protein levels in prostate cancer patients during their progression to the hormone-refractory state, which suggests that ApoC-I protein is related to progression of prostate cancer. However, as the exact role of ApoC-I in prostate cancer pathogenesis is unclear, further research is required.

Detection of nasopharyngeal carcinoma using surface-enhanced laser desorption and ionization mass spectrometry profiles of the serum proteome

Background and Objective: Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult due to the insufficient specificity of the conventional examination method. This study was to investigate potential and consistent biomarkers for NPC, particularly for early detection of NPC. Methods: A proteomic pattern was identified in a training set (134 NPC patients and 73 control individuals) using the surface-enhanced laser desorption and ionization-mass spectrometry (SELDI-MS), and used to screen the test set (44 NPC patients and 25 control individuals) to determine the screening accuracy. To confirm the accuracy, it was used to test another group of 52 NPC patients and 32 healthy individuals at 6 months later. Results: Eight proteomic biomarkers with top-scored peak mass/charge ratios (m/z) of 8605 Da, 5320 Da, 5355 Da, 5380 Da, 5336 Da, 2791 Da, 7154 Da, and 9366 Da were selected as the potential biomarkers of NPC with a sensitivity of 90.9% (40/44) and a specificity of 92.0% (23/25). The performance was better than the current diagnostic method by using the Epstein-Barr virus (EBV) capsid antigen IgA antibodies (VCA/IgA). Similar sensitivity (88.5%) and specificity (90.6%) were achieved in another group of 84 samples. Conclusion: SELDI-MS profiling might be a potential tool to identify patients with NPC, particularly at early clinical stages.

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.

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.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry traces the geographical source of Biomphalaria pfeifferi and Bulinus forskali,involved in schistosomiasis transmission

Background Freshwater snails of the genera Bulinus spp.,Biomphalaria spp.,and Oncomelania spp.are the main intermediate hosts of human and animal schistosomiasis.Identification of these snails has long been based on mor-phological and/or genomic criteria,which have their limitations.These limitations include a lack of precision for the morphological tool and cost and time for the DNA-based approach.Recently,Matrix-Assisted Laser Desorp-tion/lonization Time-Of-Flight(MALDI-TOF)mass spectrometry,a new tool used which is routinely in clinical microbi-ology,has emerged in the field of malacology for the identification of freshwater snails.This study aimed to evaluate the ability of MALDI-TOF MS to identify Biomphalaria pfeifferi and Bulinus forskali snail populations according to their geographicalorigin.Methods This study was conducted on 101 Bi.pfeifferi and 81 Bu.forskali snails collected in three distinct geo-graphical areas of Senegal(the North-East,South-East and central part of the country),and supplemented with wild and laboratory strains.Specimens which had previously been morphologically described were identified by MALDl-TOF MS[identification log score values(LSV)≥1.7],after an initial blind test using the pre-existing database.After DNA-based identification,new reference spectra of Bi.pfeiferi(n=10)and Bu.forskali(n=5)from the geographical areas were added to the MALDI-TOF spectral database.The final blind test against this updated database was per-formed to assess identification at the geographic source level.Results MALDI-TOF MS correctly identified 92.1%of 101 Bi.pfeifferi snails and 98.8%of 81 Bu.forskali snails.At the final blind test,88%of 166 specimens were correctly identified according to both their species and sampling site,with LSVs ranging from 1.74 to 2.70.The geographical source was adequately identified in 90.1%of 91 Bi.pfeifferi and 85.3%of 75 Bu.forskalii samples.Conclusions Our findings demonstrate that MALDI-TOF MS can identify and differentiate snail populations according to geographical origin.It outperforms the current DNA-based approaches in discriminating laboratory from wild strains.This inexpensive high-throughput approach is likely to further revolutionise epidemiological studies in areas which are endemic for schistosomiasis.

A Plasmonic Mass Spectrometry Approach for Detection of Small Nutrients and Toxins

Nutriology relies on advanced analytical tools to study the molecular compositions of food and provide key information on sample quality/safety. Small nutrients detection is challenging due to the high diversity and broad dynamic range of molecules in food samples, and a further issue is to track low abundance toxins. Herein, we developed a novel plasmonic matrix-assisted laser desorption/ionization mass spectrometry(MALDI MS)approach to detect small nutrients and toxins in complex biological emulsion samples. Silver nanoshells(SiO_2@-Ag) with optimized structures were used as matrices andachieved direct analysis of ~ 6 n L of human breast milk without any enrichment or separation. We performed identification and quantitation of small nutrients and toxins with limit-of-detection down to 0.4 pmol(for melamine) and reaction time shortened to minutes, which is superior to the conventional biochemical method currently in use. The developed approach contributes to the near-future application of MALDI MS in a broad field and personalized design of plasmonic materials for real-case bio-analysis.

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.

Plasmonic polydopamine-modified TiO_(2)nanotube substrates for surface-assisted laser desorption/ionization mass spectrometry imaging

Mass spectrometry imaging(MSI)has made the spatio-chemical characterization of a broad range of small-molecule metabolites within biological tissues possible.However,available matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS)suffers from severe background interferences in low-mass ranges and inhomogeneous matrix deposition.Thus,surface-assisted LDI-MS(SALDI-MS)has been an attractive alternative for high-sensitivity detection and imaging of small biomolecules.In this study,we construct a new composite substrate,hydrophobic polydopamine(hPDA)-modified TiO_(2)nanotube(TDNT)coated with plasmonic gold nanoparticle(AuNP-hPDA-TDNT),as a dual-polarity SALDI substrate using an easy and cost-effective fabrication approach.Benefitting from the synergistic effects of TDNT semiconductor and plasmonic PDA modification,this SALDI substrate exhibits superior performance for dual-polarity detection of a vast diversity of small molecules.Highly reduced background interferences,lower detection limits,and spot-to-spot repeatability can be achieved using AuNP-hPDA-TDNT substrates.Due to its unique imprinting performance,various metabolites and lipids can be visualized within jatropha integerrima petals,ginkgo leaves,strawberry fruits,and latent fingerprints.More valuably,the universality of this matrix-free substrate is demonstrated for mapping spatial distribution of lipids within mouse brain tissue sections.Considered together,this AuNP-hPDA-TDNT material is expected to be a promising SALDI substrate in various fields,especially in nanomaterial development and life sciences.

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。

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。

基于水自由基阳离子快速检测甲醇和乙醇

本文采用常压电晕放电电离质谱技术,开发了一种快速检测甲醇和乙醇含量的方法。基于水自由基阳离子分别与甲醇和乙醇相互作用形成相应的水自由基阳离子复合物[CH_(3)OH+H_(2)O]^(+·)(m/z 50)和[CH_(3)CH_(2)OH+H_(2)O]^(+·)(m/z 64),实现了不同种类酒精饮料中甲醇和乙醇的直接快速定性与定量分析。结果表明,甲醇和乙醇的检出限分别为0.011、0.004 mg/L,定量限分别为0.038、0.014 mg/L。该方法无需样品前处理、操作简单、分析速度快,适合含水体系的检测,对复杂体系中甲醇和乙醇的定量分析具有指导意义。

基于质谱的单细胞分析技术研究新进展

细胞是生物体最基本的结构和功能单位。近年来,人们对细胞异质性的关注越来越多。为了分析细胞的这种异质性,避免单细胞信息被群体细胞的平均值所淹没,单细胞分析技术不断发展起来。由于单细胞的体积小,内含物质含量低且种类繁多,某些物质在细胞内的变化迅速,分析过程中不同物质之间相互干扰,造成了单细胞分析极具挑战性。基于质谱的单细胞分析技术具有通用性强、无需标记、高灵敏度、高分辨率、高选择性的优点,已逐渐在单细胞分析技术中脱颖而出,成为单细胞分析的理想工具。本文系统地总结了近5年来基于质谱的单细胞分析技术的最新进展,包括电喷雾电离质谱、激光解吸电离质谱、二次离子质谱、电感耦合等离子体质谱,并对未来的研究和技术方向进行展望,希望为单细胞质谱分析技术的研究提供参考。

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。