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1.
用飞行质谱技术筛选结直肠癌患者中特异性生物标志物的临床意义 总被引:16,自引:5,他引:16
目的 探讨蛋白质质谱分析方法鉴定结直肠癌生物标志物的临床应用价值。方法 用美国Ciphergen Biosystems公司金属亲和表面芯片(IMAC3)和蛋白芯片仪,检测146例结直肠癌患者,62名正常人,32例结直肠良性疾病患者血清蛋白质的相对含量。结果 结直肠癌患者与正常人和结直肠良性疾病患者血清蛋白质在质荷比为4467000—15122000,其中有10个蛋白质含量差异有显著意义。146例结直肠癌患者中有142例患者被正确检测,62名正常人和32例结直肠良性疾病患者均被正确识别,检测准确率为98.3%(236/240),灵敏度和特异性分别为97.30%(142/146)和100%(94/94)。结论 该方法灵敏度和特异性高,可快速、准确检测结直肠癌,其临床应用前景广阔。 相似文献
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Michael Kiehntopf Robert Siegmund Thomas Deufel 《Clinical chemistry and laboratory medicine》2007,45(11):1435-1449
Surface-enhanced laser desorption time of flight mass spectrometry (SELDI-TOF-MS) is an important proteomic technology that is immediately available for the high throughput analysis of complex protein samples. Over the last few years, several studies have demonstrated that comparative protein profiling using SELDI-TOF-MS breaks new ground in diagnostic protein analysis particularly with regard to the identification of novel biomarkers. Importantly, researchers have acquired a better understanding also of the limitations of this technology and various pitfalls in biomarker discovery. Bearing these in mind, great emphasis must be placed on the development of rigorous standards and quality control procedures for the pre-analytical as well as the analytical phase and subsequent bioinformatics applied to analysis of the data. To avoid the risk of false-significant results studies must be designed carefully and control groups accurately selected. In addition, appropriate tools, already established for analysis of highly complex microarray data, need to be applied to protein profiling data. To validate the significance of any candidate biomarker derived from pilot studies in appropriately designed prospective multi-center studies is mandatory; reproducibility of the clinical results must be shown over time and in different diagnostic settings. SELDI-TOF-MS-based studies that are in compliance with these requirements are now required; only a few have been published so far. In the meantime, further evaluation and optimization of both technique and marker validation strategies are called for before MS-based proteomic algorithms can be translated into routine laboratory testing. 相似文献
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The urine of a 9-year-old boy suffering from extreme body odour was assayed for trimethylamine (TMA) by gas chromatography-mass spectrometry. A trapping column bed, specifically designed for volatile compounds, was used in the mass spectrometry. The diagnosis of rare trimethylaminuria was confirmed by the presence of appreciable urinary TMA. Urinary TMA concentrations were reduced to virtually nil after the elimination of the major choline sources (fish and eggs) from the diet. 相似文献
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Diamandis EP 《Clinical chemistry》2006,52(4):771-2; author reply 772
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Bouamrani A Ternier J Ratel D Benabid AL Issartel JP Brambilla E Berger F 《Clinical chemistry》2006,52(11):2103-2106
BACKGROUND: New molecular profiling technologies can aid in analysis of small pathologic samples obtained by minimally invasive biopsy and may enable the discovery of key biomarkers synergistic with anatomopathologic analysis related to prognosis, therapeutic response, and innovative target validation. Thus proteomic analysis at the histologic level in healthy and pathologic settings is a major issue in the field of clinical proteomics. METHODS: We used surface-enhanced laser desorption ionization-time-of-flight mass spectrometry (SELDI-TOF MS) technology with surface chromatographic subproteome enrichment and preservation of the spatial distribution of proteomic patterns to detect discrete modifications of protein expression. We performed in situ proteomic profiling of mouse tissue and samples of human cancer tissue, including brain and lung cancer. RESULTS: This approach permitted the discrimination of glioblastomas from oligodendrogliomas and led to the identification of 3 potential markers. CONCLUSION: Direct tissue proteomic analysis is an original application of SELDI-TOF MS technology that can expand the use of clinical proteomics as a complement to the anatomopathological diagnosis. 相似文献
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Age-related variations in acylcarnitine and free carnitine concentrations measured by tandem mass spectrometry 总被引:2,自引:0,他引:2
Cavedon CT Bourdoux P Mertens K Van Thi HV Herremans N de Laet C Goyens P 《Clinical chemistry》2005,51(4):745-752
BACKGROUND: The acylcarnitine profiles obtained from dried blood spots on "Guthrie cards" have been widely used for the diagnosis and follow-up of children suspected of carrying an inherited error of metabolism, but little attention has been paid to potential age-related variations in the reference values. In this study, we evaluated the variations in free carnitine and acylcarnitine concentrations with age, as measured by tandem mass spectrometry. METHODS: Filter-paper blood spots were collected from 433 healthy individuals over a period of 17 months. Eight age groups were defined: cord blood, 3-6 days (control group), 15-55 days, 2-18 months, 19-59 months, 5-10 years, 11-17 years, and 18-54 years. Free carnitine and acylcarnitines were measured for each individual. Mean values were calculated for each age group and compared with those for the control group. RESULTS: Free carnitine was significantly higher in older children than in newborns (P <0.05), but the concentrations of several acylcarnitines tended to be significantly lower in cord blood and in groups of older children than in the control group. Only minor sex-related differences were observed. CONCLUSION: Although the risk of underdiagnosis of fatty acid oxidation disorders with the use of newborn values as reference can be considered as small, in some circumstances the use of age-related reference values may have a potential impact on the diagnosis and management of inherited errors of metabolism. 相似文献
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BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization is robust and allows accurate measurement of both low- and high-molecular weight components of complex mixtures. We developed a LC-MS/MS method for the analysis of methylmalonic acid (MMA), a biochemical marker for inherited disorders of propionate metabolism and acquired vitamin B(12) deficiency. METHODS: We added 1 nmol of the internal standard MMA-d(3) to 500 microL of plasma or 100 microL of urine before solid-phase extraction. After elution with 18 mol/L formic acid, the eluate was evaporated, and butyl ester derivatives were prepared with 3 mol/L HCl in n-butanol at 65 degrees C for 15 min. For separation, we used a Supelcosil LC-18, 33 x 4.6 mm column with 60:40 (by volume) acetonitrile:aqueous formic acid (1 g/L) as mobile phase. The transitions m/z 231 to m/z 119 and m/z 234 to m/z 122 were used in the selected reaction monitoring mode for MMA and MMA-d(3,) respectively. The retention time of MMA was 2.2 min in a 3.0-min analysis, without interference of a physiologically more abundant isomer, succinic acid. RESULTS: Daily calibrations between 0.25 and 8.33 nmol in 0.5 mL exhibited consistent linearity and reproducibility. At a plasma concentration of 0.12 micromol/L, the signal-to-noise ratio for MMA was 40:1. The regression equation for our previous gas chromatography-mass spectrometry (GC-MS) method (y) and the LC-MS/MS method (x) was: y = 1.030 x -0.032 (S(y|x) = 1.03 micromol/L; n = 106; r = 0.994). Inter- and intraassay CVs were 3. 8-8.5% and 1.3-3.4%, respectively, at mean concentrations of 0.13, 0.25, 0.60, and 2.02 micromol/L. Mean recoveries of MMA added to plasma were 96.9% (0.25 micromol/L), 96.0% (0.60 micromol/L), and 94.8% (2.02 micromol/L). One MS/MS system used only overnight (7.5 h) replaced two GC-MS systems (30 instrument-hours/day) to run 100-150 samples per day, with reductions of total cost (supplies plus equipment), personnel, and instrument time of 59%, 14%, and 75%, respectively. CONCLUSIONS: This method is well suited for large-scale MMA testing (> or =100 samples per day) where a shorter analytical time is highly desirable. Reagents are less expensive than the anion-exchange/cyclohexanol-HCl method, and sample preparation of batches up to 100 specimens is completed in less than 8 h and is automated. 相似文献
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Anna Lee Chung Hyun Nahm JinSoo Lee MiKyeong Lee KyoungRyul Lee 《Journal of clinical laboratory analysis》2021,35(11)
BackgroundTo explore the association of thrombo‐inflammatory biomarkers with severity in coronavirus disease (COVID‐19), we measured antiphospholipid antibodies (aPL) and calprotectin in sera of COVID‐19 patients.MethodsAnticardiolipin antibodies (aCL) and anti‐β2‐glycoprotein I antibodies were measured using enzyme‐linked immunosorbent assay (ELISA) and multiplex flow immunoassay (MFIA) in hospitalized COVID‐19 patients (N = 105) and healthy controls (N = 38). Anti‐phosphatidylserine/prothrombin antibodies, calprotectin, and C‐reactive protein (CRP) levels were also measured. We assessed the potential correlation between calprotectin levels and various laboratory parameters that were measured during the hospitalization period. After stratifying COVID‐19 patients into two groups by their oxygenation status or acute respiratory distress syndrome presentation, the discriminatory performance of each biomarker was evaluated.ResultsA high proportion of COVID‐19 patients (29.5%, 31/105) had low aCL IgM titers that were detectable by ELISA but mostly below the detection limit of MFIA. Calprotectin levels in severe groups of COVID‐19 were significantly higher than those in non‐severe groups, while CRP levels revealed no significant differences. Serum calprotectin levels showed strong to moderate degree of correlation with other routinely used parameters including peak levels of CRP, ferritin, procalcitonin, BUN, and neutrophil‐to‐lymphocyte ratio, but a negative correlation with minimal lymphocyte count and CD4+ T cells. The discriminatory performance was highest for calprotectin in discriminating severe groups of COVID‐19.ConclusionsSerum calprotectin levels were significantly elevated in severe COVID‐19 cases. The prevalence of clinically significant aPL did not differ. The link between calprotectin and inflammatory pathway in COVID‐19 may help improve the management and outcomes of COVID‐19 patients. 相似文献
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利用飞行质谱法对急性心肌梗死血清标志物的研究 总被引:4,自引:0,他引:4
目的利用飞行质谱法测定的蛋白质谱改变来研究诊断急性心肌梗死(AMI)的血清标志物。方法AMI病人85例,不同时段血清120份,不稳定型心绞痛病人(UP)21例,正常对照组45例。分别将所有AMI病人血清与正常对照组比较,早期AMI病人(A1)45例与正常组比较,A1组病人与UP病人比较。利用飞行质谱法测定所有血清。结果①AMI病人与正常人之间在质荷比(M/Z)813044、814984、663782、792885、329988、430755这六种蛋白质的差异呈显著性。尤其质荷比813044的蛋白质AMI病人呈明显低表达,可作为诊断AMI的血清标志物。②A1组病人与正常人之间在质荷比814984和591000这两种蛋白质的差异呈显著性。A1组病人在质荷比814984的蛋白质呈低表达,而在质荷比591000的蛋白质呈明显高表达,可作为诊断早期AMI的血清标志物。③A1组病人与UP病人之间在质荷比173436的蛋白质差异呈显著性。A1组病人在质荷比173436的蛋白质呈低表达,可作为鉴别诊断早期AMI和UP的血清标志物。结论飞行质谱方法灵敏度和特异性高,可快速、准确诊断出早期AMI,优于TnT。 相似文献
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G Moneti G Agati M G Giovannini M Pazzagli R Salerno G Messeri M Serio 《Clinical chemistry》1985,31(1):46-49
We describe a mass-spectrometric method based on the fast atom bombardment ionization technique in the negative-ion mode for measuring pregnanediol-3 alpha-glucuronide in diluted urine from women. The procedure requires addition of testosterone-17 beta-D-glucuronide (2.5 micrograms/25 microL) to the urine sample as internal standard, and the sample is added directly to the fast atom bombardment target with no further manipulation. We have assessed and evaluated the method by the traditional criteria of reliability. 相似文献
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ObjectivesSpecific and sensitive analytical techniques to quantify therapeutic monoclonal antibodies (mAbs) are required for therapeutic drug monitoring. The quantification of mAbs has been historically performed using enzyme-linked immunosorbent assays (ELISAs), for which the limitations in terms of specificity have led to the development of alternative analytical strategies.MethodsHere, we describe the validation of a liquid chromatography tandem mass-spectrometry (LC-MS/MS) method for the simultaneous quantification of rituximab (RTX – anti-CD20) and eculizumab (ECU – anti-C5). Sample preparation was based on our previously published method, using protein G purification and trypsin digestion. A new specific peptide for RTX, containing an N-terminal pyroglutamine and a trypsin miss-cleavage, enables better sensitivity, while peptide of ECU was chosen thanks to an in silico trypsin digestion and the Skyline® software. Full-length stable-isotope-labeled adalimumab was added to plasma samples as an internal standard. RTX in 50 human serum samples was quantified by LC-MS/MS and the concentrations obtained compared to those obtained with two commercial ELISA kits (Lisa Tracker® and Promonitor®).ResultsCalibration curves were linear from 1 to 200 µg.mL−1 for RTX and 5 to 200 µg.mL−1 for ECU, and within-day and between-day accuracy and precision fulfilled Food and Drug Administration validation criteria. Comparison of the LC-MS/MS method with ELISA showed a negligible bias with the Lisa Tracker® kit (4%), but significant bias with the Promonitor® assay (mean underestimation of 69% for the Promonitor® assay).ConclusionsThis new LC-MS/MS method allows the simultaneous quantification of RTX and ECU in human samples and could be used for therapeutic drug monitoring. 相似文献
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目的:采用串联质谱标签(TMT)联合液相色谱串联质谱(LC-MS/MS)筛选免疫介导性脱髓鞘疾病诊断与鉴别诊断的潜在生物标志物。方法:选择首都医科大学附属北京天坛医院2020年1月至2021年1月收治的20例脱髓鞘疾病患者(脱髓鞘组),包括10例吉兰-巴雷综合征(GBS)患者(GBS亚组)与10例多发性硬化(MS)患者... 相似文献
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Massimo Papale Maria Carmela Pedicillo Salvatore Di Paolo Bradley John Thatcher Lorenzo Lo Muzio Pantaleo Bufo Maria Teresa Rocchetti Marta Centra Elena Ranieri Loreto Gesualdo 《Clinical chemistry and laboratory medicine》2008,46(1):89-99
BACKGROUND: Saliva is one of the most promising and easy-to-collect source of potential biomarkers of oral and systemic disease. We standardized a protocol suitable for pre-analytical treatment and for the analysis of whole normal saliva by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS). METHODS: We evaluated the impact of storage time, freeze/thaw cycles, denaturing agents, glycoproteins depletion, centrifugation, type of matrix and ProteinChip used on the quality of the SELDI protein profile. Moreover, we explored the inter-individual and between-sex differences and the changes in the sample composition over the day. RESULTS: Saliva was qualitatively stable, in the absence of protease inhibitors, for up to 3 h from the collection at room temperature, although the intensity of a number of peaks slightly decreased between 0 and 3 h and the addition of protease inhibitors did not completely revert this trend. The saliva proteome changed during the day and showed relevant between-sex differences. The protein profile remained stable for up to five freeze/thaw cycles. The addition of denaturing solutions and the depletion of glycoproteins improved the quality of the spectra without affecting their reproducibility. CONCLUSIONS: We defined a protocol that improved the quality and the reproducibility of SELDI-TOF/MS analysis, thus potentially supporting the search for putative biomarkers of disease. 相似文献
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Discovery of novel plasma biomarkers for future incident venous thromboembolism by untargeted synchronous precursor selection mass spectrometry proteomics
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S. B. Jensen K. Hindberg T. Solomon E. N. Smith J. D. Lapek Jr D. J. Gonzalez N. Latysheva K. A. Frazer S. K. Brækkan J.‐B. Hansen 《Journal of thrombosis and haemostasis》2018,16(9):1763-1774
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Robert M Kok Desirée E C Smith Rob Barto Annemieke M W Spijkerman Tom Teerlink Henk J Gellekink Cornelis Jakobs Yvo M Smulders 《Clinical chemistry and laboratory medicine》2007,45(7):903-911
BACKGROUND: Alterations in global DNA methylation are implicated in various pathobiological processes. We describe a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for determination of cytosine and 5-methylcytosine in DNA. METHODS: DNA was hydrolyzed using formic acid. Cytosine and 5-methylcytosine were separated by gradient-elution reversed-phase chromatography with a mobile phase containing nonafluoropentanoic acid (NFPA) as ion-pairing reagent and quantified using stable isotope dilution LC-ESI-MS/MS. The method was applied to DNA isolated from leukocytes of healthy volunteers. RESULTS: Linear calibration curves were obtained in the range 0.111-4.422 ng/microL [mean correlation co-efficient 0.9983 (SD=0.0011), n=9] for cytosine and 0.0048-0.1936 ng/microL [mean correlation coefficient 0.9991 (SD=0.0010), n=9] for 5-methylcytosine. The intra- and inter-assay CVs for the 5-methylcytosine/total cytosine ratio (mCyt/tCyt) was 1.7% (n=9) and 3.5% (n=8) for calf thymus DNA (mean mCyt/tCyt ratio 6.5%), and 4.5% (n=6) and 6.5% (n=14), respectively for pBR322 DNA (mean mCyt/tCyt ratio 0.48%). The limit of detection (signal-to-noise ratio 3) was 2 pg on-column for cytosine and 5-methylcytosine. In healthy subjects (n=109), the mCyt/tCyt ratio varied from 2.6% to 4.8% (median 4.1%). DNA methylation was negatively correlated to age, but only in subjects with the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype (p=0.046). No association with B-vitamin status was observed. CONCLUSIONS: This LC-ESI-MS/MS method is easy to perform and offers reproducibility, selectivity and sensitivity for studying DNA methylation. The method allows a sample throughput of approximately 200 samples/week. The MTHFR C677T genotype influences age-related changes in DNA methylation. 相似文献