New serum biomarkers for detection of endometriosis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This study used proteomic fingerprint technology, combining nano-sized magnetic beads with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), to screen for potential protein biomarkers for the diagnosis of endometriosis. Serum proteins from 126 patients with endometriosis and 120 healthy controls were profiled and compared. Biomarker pattern software identified 46 discriminating mass-to-charge m/z ratio peaks that were related to endometriosis. The model constructed by the software, based on three of these peaks (m/z 5988.7, 7185.3 and 8929.8), generated excellent separation between the endometriosis and control groups. The sensitivity was 91.4% and the specificity 95.0%. Blind testing on a second series of serum samples from patients with endometriosis and healthy controls indicated a sensitivity of 89.3% and a specificity of 90.0%. Biomarkers for endometriosis can be discovered in serum by MALDI-TOF-MS in combination with nano-sized magnetic beads. The pattern of combined markers provides a powerful and reliable diagnostic method for endometriosis, with high sensitivity and specificity.     

基于激光解析／离子化-飞行时间质谱技术的中药阿胶蛋白质组分析

目的:应用激光解析/离子化-飞行时间质谱技术对传统中药阿胶蛋白/肽成分进行蛋白质组初步分析,建立阿胶蛋白质质量指纹图。方法:实验于2004-06/2005-03在湖南中医药大学蛋白质组学实验室完成。采用饱和硫酸胺沉淀,透析脱盐冻干法获得阿胶水溶性蛋白/肽,采用Ciphergen BiosystemsInc.ProteinChipRBiology System(美国PBSⅡ )及配套的NP10芯片、激光解析/离子化-飞行时间质谱技术,分析阿胶Mr1500～13000区间蛋白质、肽分布及其相对分子质量。结果:Mr2000～4000区间显示4个相对分子质量峰,可能是2种肽;Mr4000～5000区间显示8个相对分子质量峰,可能为2种肽;Mr5000～5500区间显示16个相对分子质量峰,约为3种肽;Mr5500￣6200区间无峰;Mr8100～8200区间显示5个相对分子质量峰,可能为1种肽;Mr11200～11400区间显示5个相对分子质量峰,可能为1种蛋白质。不同质量浓度稳定获得的有意义蛋白/肽共计9个。不同相对分子质量肽之间差分别提示可能为丙氨酸、丝氨酸、苯丙氨酸残基相对分子质量。结论:通过分析,可以形成阿胶蛋白质/肽成分质量指纹图,可作为阿胶数字化质控标准;并为进一步分离、纯化及验证阿胶功能相关活性蛋白质/肽组分提供可靠信息。     

A rapid test for the diagnosis of thrombotic thrombocytopenic purpura using surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF)-mass spectrometry

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP), a life-threatening thrombotic microangiopathy, requires immediate diagnosis and plasma exchange therapy. Development of TTP is related to functional deficiency of ADAMTS-13 protease that leads to the accumulation of ultra large von Willebrand factor (VWF) and subsequent platelet thrombosis. Currently no clinical test is available for the rapid detection of ADAMTS-13 activity. OBJECTIVES: The goal is to devise a novel method to rapidly detect functional activity of ADAMTS-13 and improve clinical outcome. METHODS AND RESULTS: A recombinant VWF substrate containing the ADAMTS-13 cleavage site and a 6X Histidine tag was cleaved by ADAMTS-13 in a dose-dependent manner, generating approximately 7739 Da peptide containing a 6X Histidine tag. This cleaved peptide, bound to an IMAC/Nickel ProteinChip, was quantified using Surface Enhanced Laser Desorption/Ionization Time-of-flight Mass Spectrometry (SELDI-TOF-MS). The assay is capable of quantifying ADAMTS-13 activity as low as 2.5% in plasma within 4 h. When the cleaved peptide was quantified as a ratio of an internal control peptide, the test displayed good reproducibility, with an average inter-assay coefficient of variation (CV) of < 33%. Further validation revealed a mean ADAMTS-13 activity of 92.5% +/- 16.6% in 39 healthy donors. Sixteen patients with idiopathic TTP displayed mean ADAMTS-13 activity of 1.73% +/- 3.62%. Further utility of this novel method includes determining the inhibitory titer of ADAMTS-13 antibody in cases of acquired TTP. CONCLUSIONS: We have devised a novel SELDI-TOF-MS assay that offers a rapid, cost-effective, and functionally relevant test for timely diagnosis and management of TTP.     

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in Alzheimer's disease

Alzheimer's disease is becoming an increasing problem in our aging society. According to our knowledge, so far, no effective pharmacotherapy to cure the cause of the disease has been developed. Therefore, early diagnosis is needed, which will result in implementation of a drug therapy aimed at decreasing and/or inhibiting disease development. Mass spectrometry techniques (MS) have a wide range of applications in proteomics and the search for biomarkers of neurodegenerative disorders, opening new possibilities in diagnostics. Identification of proteins in body fluids (like cerebrospinal fluid or blood) is possible due to MS spectra analysis. The detected changes in protein concentrations are connected with pathological states in an organism and, therefore, can be regarded as biomarkers. Developing procedures for proteome analysis might result in fast diagnosis, as well as creating better suited pharmaceuticals. This paper reviews the search of biomarkers in cerebrospinal fluid and blood. Later on, the use of matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in proteomics, focusing on blood-related biomarkers, is discussed. The aim of the work is also to highlight the advantages and disadvantages of MALDI-TOF-based analyses.     

Analysis of minor hemoglobins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

BACKGROUND: Hemoglobin (Hb) heterogeneity arises mainly from posttranslational modifications of the globin chains, and cation-exchange chromatography reveals falsely increased concentrations of some minor Hbs in the presence of abnormal Hbs. Here we describe a method for identification of the globin chains and their posttranslational modifications contained in the Hb fractions. METHODS: We used cation-exchange HPLC (PolyCAT A column) for separation of Hb fractions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for analysis of the separated globin chains. Globin chains were identified by their molecular masses. Posttranslational modifications of globin chains were identified by digestion of the proteins with endoproteinase V8 before MALDI-TOF MS of the resulting peptides. RESULTS: Analysis of the HbA2 fractions of patients with HbS revealed 4 different globin chains. We found, in addition to the expected alpha- and delta-chains, the carbamylated alpha- and the betaS-chains. Additionally, we analyzed HbH, Hb Barts, HbA 1b, pre-HbA 1c, HbA 1c, HbF1, HbF, HbA 1d3a, HbA 1d3b, HbA2, and HbC1 fractions from control and pathologic blood samples. We identified several posttranslational modifications of the globin chains, such as pyruvatization, glycation, acetylation, carbamylation, and acetaldehyde adduct formation. CONCLUSIONS: The native and posttranslationally modified globin chains in minor and major Hbs are unambiguously identified by MALDI-TOF MS. A minor Hb containing the carbamylated alpha- and the betaS-chain elutes at the same time as normal HbA2 (alpha2delta2) and thus leads to falsely increased HbA2 values in patients with HbS when blood is analyzed with PolyCAT A chromatography.     

飞行时间质谱检测HBV耐药位点变异及其影响因素探讨

目的 建立PCR-MALDI-TOF MS检测乙型肝炎病毒针对核苷类药物耐药突变位点的检测方法,探讨PCR-MALDI-TOF MS技术的影响因素,为正确应用该技术提供参考.方法 采用PCR-MALDI-TOF MS检测10份HBV质粒标准品及100份HBV阳性临床标本(均单独或者联合使用过拉米夫定、阿德福韦酯、恩替卡韦和替比夫定等核苷类药物),并用PCR产物测序进行验证.结果 100份HBV阳性的临床标本,PCR-MALDI-TOF MS检测耐药阳性结果31份,阴性结果69份.与测序结果相比完全一致的有94份(94%),6份检测结果不一致,其中3份质谱与测序结果均为耐药阳性,但质谱检测结果耐药位点多于测序.PCR-MALDI-TOF MS检测灵敏度为100 copies/μl,突变型的检出阈值为5%.结论 PCR-MALDI-TOF MS技术检测HBV耐药位点变异具有高灵敏度,高准确率,高通量和自动化的特点,适合用于临床对HBV耐药位点进行基因检测.

Abstract：

Objective To establish a rapid method for detection of drug-resistance mutation in HBV, based on PCR-MALDI-TOF MS, and to explore the influential factors on this method. Methods One hundred blood serum samples, which were collected from chronic HBV patients with single drug-resistance or multiple drug-resistance of Lamivudin, Adefovi, Entecavir and Telbivudine, and 10 kinds of mutant HBV plasmids were analyzed using PCR-MALDI-TOF MS and confirmed by PCR-based sequencing. Results Of 100 samples detected, thirty-one samples were positive for drug-resistance and 69 samples were negative. The PCR-MALDI-TOF MS results of 94 samples were completely consistent with PCR-based sequencing. Six samples were inconsistent , of which three samples were positive by the two methods, but more mutation loci were detected by PCR-MALDI-TOF MS than sequencing. The consistent rate of two methods was 94%,detection sensitivity was up to 100 copies/μl, and the cut off value of detectable mutation level was 5%.Conclusion PCR-MALDI-TOF MS could be used for rapid and simple analysis of the drug resistance for the clinical application with features of high sensitivity and accuracy, high throughput and automation.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for genotyping of human platelet-specific antigens

BACKGROUND: Genotyping of single-nucleotide polymorphisms (SNPs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique, where finally tools for end users have become available to design primers and analyze SNPs of their own interest. This study investigated the potential of this technique in platelet (PLT) genotyping and developed a validated method for genotyping of clinical relevant human PLT antigens (HPAs).
STUDY DESIGN AND METHODS: A multiplex assay using MALDI-TOF MS to analyze six HPA loci (HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, and HPA-15) simultaneously in a single reaction was applied for the genotyping of 100 DNA samples from a cohort of plateletpheresis donors and a patient population (n = 20) enriched for rare alleles. The genotyping results using MALDI-TOF MS were validated by the comparison with the results from typing by polymerase chain reaction with sequence-specific primers and conventional DNA sequencing.
RESULTS: Both homozygous and heterozygous genotypes of HPA-1 to -5 and -15 of the 120 individuals were easily identified by a six-plexed assay on MALDI-TOF MS. The three approaches achieved a 100 percent concordance for the genotyping results of the six HPA loci.
CONCLUSION: Compared to conventional methods, the MALDI-TOF MS showed several advantages, such as a high velocity, the ability to perform multiplexed assays in a single reaction, and automated high-throughput analysis of samples. This enables cost-efficient large-scale PLT genotyping for clinical applications.     

表面增强激光解析电离飞行时间质谱技术的质量控制与标准化

表面增强激光解析电离飞行时间质谱（SELDI-TOF-Ms）技术是近年兴起的蛋白质组学研究前沿技术，其标准化和质量控制是其顺利应用到临床的关键。我们对SELDI-TOF-MS检测样本采集与制备、试剂的选择与保存、实验仪器的校准与维护；实验结果重复性验证与分析；数据处理与分析的标准化方法的选择、SELDI－TOF-MS技术系统标准化进行探讨和评价。     

Identification of lethal Aspergillus at early growth stages based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Delayed and incorrect diagnoses are potential risk factors leading to high mortality of invasive aspergillosis (IA). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to acquire a wide mass spectral range and characterize the early process of asexual sporulation of lethal IA pathogens recovered on agar plates. Proteins were extracted using trifluoroacetic acid and soft ionized using an ultraviolet laser with the assistance of ferulic acid. At the second stage of sporulation with various differentiated structures, there are more specific peaks that can be used to discriminate different Aspergillus species than at the first stage, which features vegetative hyphae. Certain specific peaks are found in different strains of the same species, Aspergillus fumigatus. In addition, the relative standard deviations of the m/z ratios are much smaller than those of the relative intensities in these peaks. Therefore, common lethal Aspergillus species can be identified after short-term cultivation by matching species-specific m/z values.     

Optimization and evaluation of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) with reversed-phase protein arrays for protein profiling.

Surface-enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry with protein arrays has facilitated the discovery of disease-specific protein profiles in serum. Such results raise hopes that protein profiles may become a powerful diagnostic tool. To this end, reliable and reproducible protein profiles need to be generated from many samples, accurate mass peak heights are necessary, and the experimental variation of the profiles must be known. We adapted the entire processing of protein arrays to a robotics system, thus improving the intra-assay coefficients of variation (CVs) from 45.1% to 27.8% (p<0.001). In addition, we assessed up to 16 technical replicates, and demonstrated that analysis of 2-4 replicates significantly increases the reliability of the protein profiles. A recent report on limited long-term reproducibility seemed to concord with our initial inter-assay CVs, which varied widely and reached up to 56.7%. However, we discovered that the inter-assay CV is strongly dependent on the drying time before application of the matrix molecule. Therefore, we devised a standardized drying process and demonstrated that our optimized SELDI procedure generates reliable and long-term reproducible protein profiles with CVs ranging from 25.7% to 32.6%, depending on the signal-to-noise ratio threshold used.     

MALDI-TOF MS技术在鲍曼不动杆菌鉴定及同源性分析中的应用

目的评价基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF MS)技术对我院鲍曼不动杆菌属的鉴定及同源性分析的能力。方法对2014年9月至2015年2月我院29株经PhoneixTM100全自动微生物鉴定仪鉴定为鲍曼不动杆菌属的菌株进行MALDI-TOF MS鉴定,用MALDI-Biotyper软件进行同源性分析,并用16S rRNA基因测序进行验证。结果 MALDI-TOF MS鉴定结果为鲍曼不动杆菌(Acinetobacter baumannii)26株,院内不动杆菌(Acinetobacter nosocomialis)3株。MALDI-TOF MS鉴定为院内不动杆菌的3株菌株的16S rRNA基因测序结果显示,2株为院内不动杆菌,1株为鲍曼不动杆菌。26株质谱鉴定为鲍曼不动杆菌的菌株分为2大簇(Ⅰ型,Ⅱ型),Ⅱ型又分为2小簇(Ⅱa、Ⅱb)。Ⅰ型、Ⅱ型分布在我院4个病区。结论MALDI-TOF MS技术鉴定鲍曼不动杆菌精准,可以鉴别鲍曼不动杆菌和院内不动杆菌。MALDI-Biotyper数据库软件进行同源性分析十分快捷。     

感染性疾病多学科交叉研究专题基质辅助激光解析电离飞行时间质谱技术在病原菌鉴定中的应用

目的 探讨基质辅助激光解析电离飞行时间质谱技术(MALDI-TOF MS)在快速鉴定血流感染病原菌的应用价值,并分析MALDI-TOF MS方法鉴定病原菌的准确性.方法 收集321例血流感染患者的血液标本进行血培养,微生物室细菌培养结果均为阳性.从血培养阳性瓶中取病原菌至分离胶促凝管中,应用MALDI-TOF MS对富...     

基于基质辅助激光解吸电离飞行时间质谱的克柔念珠菌遗传分化研究

目的在蛋白质水平明确克柔念珠菌的遗传分化特征，建立基于基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）的不同谱系克柔念珠菌的鉴定方法。方法选取20株克柔念珠菌，采用MALDI-TOF MS结合分析软件ClinProTools，确定克柔念珠菌不同谱系的质谱标志峰，构建基于遗传算法（GA）的分类模型，用于区分不同谱系的克柔念珠菌菌株，选择25株克柔念珠菌对该模型进行验证。结果利用20株克柔念珠菌构建的GA分类模型，反映模型正确鉴别能力的识别能力值（RC）为100%，反映模型处理测试谱图变异能力的交叉验证值（CV）为97.89%。 25株克柔念珠菌、150张谱图外部验证模型显示其分类能力为95.30%。 确定13个克柔念珠菌谱系相关的特征峰（m/z 3 971.40、3 136.95、3 427.33、2 405.28、2 996.73、2 913.95、3 376.97、6 736.13、5 819.03、4 045.16、5 869.00、3 618.10及3 946.14），可用于正确区分来自不同谱系的克柔念珠菌。结论本研究论证并确认了克柔念珠菌的群体遗传分化事件，同时也建立了一种简便而有效的快速谱系分型方法。     

基质辅助激光解析飞行时间质谱在临床病原菌检测中的应用进展

近年来质谱仪技术有了快速的发展.20世纪80年代,基质辅助激光解析飞行时间质谱( matrix assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF-MS)的诞生,奠定了基于蛋白质指纹图谱鉴定微生物的硬件基础.每种微生物都有自身独特的蛋白质组成,质谱仪具有测定荷电离子质量的能力,可测得待检微生物的蛋白质质量指纹图谱,经软件对这些指纹图谱进行处理并和数据库中各种已知微生物的标准指纹图谱进行比对,即可以完成对微生物的鉴定.由于测定的图谱中主要的分子离子峰为菌体内高丰度,且表达稳定、进化保守的核糖体蛋白,因此这一方法不仅鉴定结果可靠,而且还可以通过聚类分析获得微生物间的进化和亲缘关系.     

Matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry to detect emerging pathogenic Candida species

Matrix-assisted laser desorption/ionization time-of-flight intact cell mass spectrometry (MALDI-TOF-ICMS) was used to differentiate pathogenic Candida species, difficult to identify by traditional methods such as growth and biochemical reactions. Results showed that species complexes like C. parapsilosis, C. orthopsilosis, and C. metapsilsosis, and very closely related species like C. glabrata and C. bracarensis, and C. albicans and C. dubliniensis could be clearly separated. MALDI-TOF-ICMS stands out as a promising tool for the rapid detection of emerging pathogens.     

用MALDI-TOF MS技术快速鉴定临床分离棒状杆菌属细菌

目的评价基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF MS)快速鉴定临床分离的非白喉棒状杆菌的价值。方法收集本院58株非白喉棒状杆菌,用MALDI-TOF MS和16S rRNA基因测序两种方法进行鉴定和比较。用MALDI-Biotyper软件构建不同棒状杆菌MALDI-TOF MS蛋白系统发育树。结果 58株非白喉棒状杆菌中,54株MALDI-TOF MS与16S rRNA基因测序鉴定结果一致,包括34株纹带棒状杆菌(Corynebacterium straitum)、11株杰氏棒状杆菌(C.jeikeium)、3株C.resistens、2株解葡萄糖苷棒状杆菌(C.glucuronolyticum)、2株黏金色棒状杆菌(C.aurimucosum)和2株无枝菌酸棒状杆菌(C.amycolatum)。4株16S rRNA基因测序无法鉴定到种的菌株中,MALDI-TOF MS鉴定为2株产黏棒状杆菌(C.mucifaciens)、1株C.singulare和1株假白喉棒状杆菌(C.commune)。结论 MALDI-TOF MS可将棒状杆菌属细菌快速、准确地鉴定到种。     

Characterization of glycation adducts on human serum albumin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

BACKGROUND: Non-enzymatic glycation of human serum albumin (HSA) is associated with the long-term complications of diabetes. We examined the structure and location of modifications on minimally-glycated HSA and considered their possible impact on the binding of drugs to this protein. METHODS: Minimally-glycated and normal HSA (used as a control) were digested with trypsin, Glu-C or Lys-C, followed by fractionation of the resulting peptides and their analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to determine the structures and locations of glycation adducts. RESULTS: Several specific lysine and arginine residues were identified as modification sites in minimally-glycated HSA. Residues K12, K51, K199, K205, K439 and K538 were found to be modified through the formation of fructosyl-lysine, while the modification of K159 and K286 involved the formation of pyrraline or N(epsilon)-carboxymethyl-lysine, respectively. Lysine K378 was found to give N(epsilon)-carboxyethyl-lysine in some forms of glycated HSA but fructosyl-lysine in other forms. Residues R160 and R472 produced a modification based on N(epsilon)-(5-hydro-4-imidazolon-2-yl)ornithine. Lysine R222 was modified to produce argpyrimidine, N(epsilon)-[5-(2,3,4-trihydroxybutyl)-5-hydro-4-imidazolon-2-yl]ornithine or tetrahydropyrimidine. CONCLUSIONS: With the exception of K12, K199, K378, K439 and K525, all of the observed sites of modification for minimally-glycated HSA were new to this current study. The fact that many of these glycation-related modifications are located at or near known drug binding sites on HSA explains why some differences have been previously noted in the binding of certain drugs to normal vs glycated HSA.