Direct-tissue SELDI-TOF mass spectrometry analysis: a new application for clinical proteomics

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.     

用基质辅助激光解吸电离飞行时间质谱进行血清蛋白质组学分析中样品稳定性研究

目的 探索利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF Ms)技术,进行血清样品中蛋白质组学研究的稳定性和重复性.方法 采用德国布鲁克公司Microflex型号质谱仪,应用线性模式,采集范围800～10 000 m/z,以样品前处理(MB-wcx)试剂盒预处理样品,CLNPROT(TM)系统的FlexAnalysis和ClinproTools软件进行数据分析.结果 本研究是在方法学已经建立的基础上,对样品稳定性进行研究.分别对随机选取的2例肿瘤患者和1名健康人群血清样本进行的平行实验结果分析表明,在本实验条件下,所测样品的质荷比(即m/z比值)漂移在实验误差允许范围内,其变异系数(CV值)分别为12.7%、13.1%和18.8%,能够明确区分疾病与健康样本;同时扩大样本量对30名健康人进行验证,其整体峰型和特征性峰保持一致,平均CV值为25.5%,说明其稳定性和重复性完伞满足临床医学实验窒分析要求.结论 在已优化的实验条件下,利用生物质谱技术可获得稳定的血清多肽谱,具有潜在辅助诊断疾病价值.     

Ion suppression in mass spectrometry

BACKGROUND: Mass spectrometry (MS) is being introduced into a large number of clinical laboratories. It provides specificity because of its ability to monitor selected mass ions, sensitivity because of the enhanced signal-to-noise ratio, and speed because it can help avoid the need for intensive sample cleanup and long analysis times. However, MS is not without problems related to interference, especially through ion suppression effects. Ion suppression results from the presence of less volatile compounds that can change the efficiency of droplet formation or droplet evaporation, which in turn affects the amount of charged ion in the gas phase that ultimately reaches the detector. CONTENT: This review discusses materials shown to cause ion suppression, including salts, ion-pairing agents, endogenous compounds, drugs, metabolites, and proteins. Experimental protocols for examining ion suppression, which should include, at a minimum, signal recovery studies using specimen extracts with added analyte, are also discussed, and a more comprehensive approach is presented that uses postcolumn infusion of the analyte to evaluate protracted ionization effects. Finally, this review presents options for minimizing or correcting ion suppression, which include enhanced specimen cleanup, chromatographic changes, reagent modifications, and effective internal standardization. SUMMARY: Whenever mass spectrometric assays are developed, ion suppression studies should be performed using expected physiologic concentrations of the analyte under investigation.     

基于质谱的蛋白质组学诊断所面临的挑战

过去的几年中,基于质谱技术的蛋白质组学分析方法已经成为寻找新的疾病标志物的重要手段之一.由于它具有高灵敏度、高通量、快速以及能和生物信息学技术结合同时快速分析大量的蛋白质或多肽的优点,使之成为临床研究的有力工具.然而,最近的一些研究显示,因为这项技术的重复性和数据统计分析等方面的不足而受到了争议.本文介绍了采用该方法进行标志物筛查时在实验设计、质谱技术以及数据分析等方面面临的一些挑战,探讨其在血清或血浆分析中的应用.     

Label-free mass spectrometry-based proteomics for biomarker discovery and validation

For most diseases, better biomarkers are urgently needed to enable (early) detection, diagnosis, prognosis, stratification for therapy and response monitoring. Proteomics delineates gene products that carry out the majority of cellular functions, and thereby may not only yield insight into altered signaling pathways in disease, but also yield novel biomarkers. In recent years, great progress has been made in mass spectrometry-based analysis of clinical tissues and biofluids, with identification and quantification of thousands of proteins now becoming increasingly routine. However, biomarker validation and clinical translation has turned out to be challenging. In this review, we summarize current mass spectrometry-based proteomics strategies for biomarker discovery and verification using selected reaction monitoring, with a focus on progress and recent applications in clinical material using label-free approaches.     

Pitfalls with mass spectrometry in clinical anesthesia

Mass spectrometry of respired gases puts a powerful analytic tool into the hand of the clinician. However, serious misinterpretations may result if the principle of operation and certain weaknesses of spectrometry are not appreciated. The potential pitfalls of clinical mass spectrometry are related to the need to have one unit serve many patients and to the design of the spectrometer and its algorithms.     

Reproducibility in protein profiling by MALDI-TOF mass spectrometry

BACKGROUND: Protein profiling with high-throughput sample preparation and MALDI-TOF MS analysis is a new potential tool for diagnosis of human diseases. However, analytical reproducibility is a significant challenge in MALDI protein profiling. This minireview summarizes studies of reproducibility of MALDI protein profiling and current approaches to improve its analytical performance. METHODS: The PubMed database was searched using combinations of the following search terms: MALDI, SELDI, reproducibility, variation, precision, peak intensity, quantification, peptide, biomarkers, and proteomics. Acceptance criteria were detailed reports on the reproducibility with MALDI protein profiling and studies describing efforts to improve the analytical performance with this technology. RESULTS: The reported intraexperiment CVs of the peak intensity vary highly between individual protein peaks, with the reported mean CV of the peak intensity varying among studies from 4% to 26%. There is additional interexperiment variation in peak intensity. Current approaches to improve the analytical performance of MALDI protein profiling include automated sample processing, extensive prefractionation strategies, immunocapture, prestructured target surfaces, standardized matrix (co)crystallization, improved MALDI-TOF MS instrument components, internal standard peptides, quality-control samples, replicate measurements, and algorithms for normalization and peak detection. CONCLUSIONS: Further evaluation and optimization of MALDI-TOF MS is recommended before use in routine analysis.     

Tandem mass spectrometry in the clinical chemistry laboratory

Tandem mass spectrometry is becoming an increasingly important analytical technology in the clinical laboratory environment. Applications in toxicology and therapeutic drug monitoring have opened the door for tandem mass spectrometry and now we are seeing a vast array of new applications being developed. It has been the combination of tandem mass spectrometry with sample introduction techniques employing atmospheric pressure ionization that has enabled this technology to be readily implemented in the clinical laboratory. Although its major research applications started with pharmacology and proteomics, tandem mass spectrometry is being used for a great variety of analyses from steroids to catecholamines to peptides. As with chromatographic methods, tandem mass spectrometry is most cost effective when groups of compounds need to be measured simultaneously. However as the price/performance of this technology continues to improve, it will become even more widely utilized for clinical laboratory applications.     

Clearing drug interferences in myeloma treatment using mass spectrometry

ObjectiveTo explore the possibility of using a combination of a rapid MALDI-TOF-MS method (Mass-Fix) in conjunction with higher resolution LC-ESI-QTOF-MS (miRAMM) measurements to discriminate the IgG kappa M-protein from daratumumab, elotuzumab and isatuximab in myeloma patients.Design & Methods86 patients with an IgG kappa M-protein were spiked with therapeutic levels of the drugs and examined by Mass-Fix and miRAMM to establish the percent of cases that could be resolved by each method. The method was then applied to 21 samples from patients receiving one of the drugs.ResultsMass-Fix was capable of resolving the t-mAb from M-protein for 87 percent of the spiked samples. For the cases unresolved by Mass-Fix, miRAMM was capable of resolving the remaining drug interferences. The 21 IgG kappa myeloma patients that were receiving the drugs were all resolved by Mass-Fix.ConclusionThis proposed algorithm allows use of a clinical available assay (Mass-Fix) while maximizing the number of cases that can accurately resolve the t-mAb from the M-protein.     

Thyroid hormone testing by tandem mass spectrometry

In the euthyroid person, absolute free thyroxine concentrations remain constant and correlate with the tissue hormone level, its biologic effect and the metabolic status of the patient. However, most circulating thyroid hormone is bound to plasma proteins and only a minute amount is in the unbound free form. Studies have shown that current free thyroxine immunoassays are binding protein dependent. Novel high-performance liquid chromatography tandem mass spectrometry (LC/MS/MS) methods have successfully dealt with problems inherent in many immunoassays for thyroid hormones and afforded improved specificity and accuracy in thyroid hormone measurements.We emphasize problems with thyroid hormone testing employing immunoassays including direct and indirect thyroid hormone immunoassays, sample processing, methods of free hormone separation and review the emerging role of liquid chromatography-tandem mass spectrometry in thyroid hormone testing. The latest generation of tandem mass spectrometers has superior limits of quantification, permitting omission of previously employed derivatization steps.Liquid chromatography-tandem mass spectrometry affords the specificity, precision, and limits of quantification necessary for the reliable measurement of thyroid hormones, enhancing diagnostic capabilities, and affording the profiles of the iodothyronines and thyronamines. These methods are especially important in states of disease and during pregnancy when protein binding is a factor that interferes with other methods for thyroid hormone analysis.     

Determination of creatinine in urine by tandem mass spectrometry

BACKGROUND: Electrospray tandem mass spectrometry (TMS) is a very powerful tool that enables one to perform high sample throughput analysis. This paper describes a method to determine creatinine in urine by tandem mass spectrometry with direct sample infusion into an ion source. METHODS: Samples (50 microl) were diluted with internal standard (IS) (450 microl of 0.667 mmol/l deuterated creatinine). Diluted samples were introduced into mass spectrometer with no prior pretreatment and after purification on ion-exchange 96-column cartridge. Tandem mass spectrometry analyses were performed in selected reaction monitoring mode. Creatinine and creatinine-d(3) were monitored using precursor and product ion settings (m/z 114 to 86 and m/z 117 to 89, respectively). The time of an analysis was 3.015 min. Both TMS methods were compared mutually and with the results obtained by enzymatic and Jaffe method. RESULTS: Linearity was obtained in the range 0.06-60 mmol/l. Detection limit was 0.2 mumol/l and recoveries were in the range 95.1-98.3% for both the assays with and without ion-exchange column. Results of both assays are in good agreement with those obtained by enzymatic and Jaffe method based on log-transformed Bland-Altman plots. Electrospray tandem mass spectrometry method utilizing both approaches with and without ion-exchange column is acceptable according to CLIA criteria. CONCLUSION: Tandem mass spectrometry allows rapid, sensitive and selective determination of creatinine in untreated urine.     

The screening of inborn errors of metabolism in sick Chinese infants by tandem mass spectrometry and gas chromatography/mass spectrometry

BackgroundAnalyses of amino acid/acylcarnitines in dried blood spots (DBS) and organic acids in urine are the primary tests for inborn errors of metabolism (IEMs). Automated tandem mass spectrometry (MS/MS) and gas chromatography/mass spectrometry (GC/MS) can rapidly and simultaneously detect numerous metabolic compounds with high precision and sensitivity.MethodsThree thousand four hundred and twenty-nine DBSs and 2781 urine samples were collected from our hospital patients with suspected IEMs, and analyzed for amino acid/acylcarnitines and organic acids by MS/MS and GC/MS, respectively. The results were used in a coincidental survey to determine the efficacy of these methods for the diagnosis of IEMs.ResultsNineteen different types of IEMs were detected in 121 affected cases (1.95% of 6210 samples). There were 66.12% amino acid disorders, 29.75% organic acid disorders and 4.13% with fatty acid oxidation disorders. Conclusions: the sick infants tested in this study had high prevalence rates of neonatal intrahepatic cholestasis, methylmalonic acidemia, hyperphenylalaninemia, tyrosinemia type I, and urea cycle disorders.ConclusionThe combined use of MS/MS and GC/MS is an appropriate tool for screening of IEMs in sick infants.     

Methanol-associated matrix effects in electrospray ionization tandem mass spectrometry

BACKGROUND: Matrix effects can profoundly reduce the performance of electrospray ionization mass spectrometry. Preliminary observations indicated that the methanol used in the mobile phase could be a source of differential ionization or ion suppression. METHODS: Drug stability studies, analysis of biological extracts, mixing experiments, and postcolumn infusions were used to test 9 commercial methanols for ionization differences in liquid chromatography-tandem mass spectrometry assays for immunosuppressants. Area responses for the drugs and internal standards were compared for mobile phases prepared with each selected methanol. Postcolumn infusion experiments were performed to confirm the degree of ionization differences occurring at the ion source, and to evaluate the proportions of ammonium, sodium, and potassium adducts. RESULTS: The decrease in signal for the immunosuppressant drugs was shown to result from differential ionization associated with the selected methanols. Product ion intensity varied by 10-fold among the methanols tested. For sirolimus, tacrolimus, and mycophenolic acid, the percentage change in ionization was the same for the drug and its corresponding internal standard. Postcolumn sirolimus infusion evaluation revealed that a 1000-fold analyte concentration difference did not affect ionization. The proportions of ammonium, sodium, and potassium adducts of sirolimus precursor ions differed in relation to the source of methanol. CONCLUSIONS: Organic solvents used in mobile phases and extract preparation of biological samples may be associated with ion suppression, affecting adduct formation and assay sensitivity.     

Dipalmitoylphosphatidylcholine in amniotic fluid quantified by fast-atom-bombardment mass spectrometry

Dipalmitoylphosphatidylcholine (DPC) is quantified by taking advantage of stable-isotope-labeled d9-DPC as internal standard. Use of a mass spectrometer to measure the ratio of d9/d0 makes this procedure a quantitative one. d9-DPC was synthesized by refluxing dipalmitoylethanolamine with d3-methyl iodide in methanol in the presence of sodium bicarbonate for 26 h. The yield of d9-phosphatidylcholine (d9-lecithin) was 89% after column-chromatographic purification. Fast atom bombardment was used to desorb the preformed phosphatidylcholine ions in a mass spectrometer of Nier-Johnson geometry. In our assessment of accuracy and precision of this technique, we found a correlation coefficient of 0.9994 between signal and sample concentration. The method was less precise when the total d0- plus d9-DPC was less than 0.2 micrograms or when the ratio of d0- to d9-lecithin exceeded 100. The within-run CV was about 1.0%. The amount of DPC in amniotic fluid samples assessed by mass spectrometry was compared with results for total phosphatidylcholine quantified by thin-layer chromatography. The fate of DPC in various laboratory manipulations was also studied.