Determination of peanut allergens in cereal-chocolate-based snacks: metal-tag inductively coupled plasma mass spectrometry immunoassay versus liquid chromatography/electrospray ionization tandem mass spectrometry

A comparison of two methods for the identification and determination of peanut allergens based on europium (Eu)-tagged inductively coupled plasma mass spectrometry (ICP-MS) immunoassay and on liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with a triple quadrupole mass analyzer was carried out on a complex food matrix like a chocolate rice crispy-based snack. The LC/MS/MS method was based on the determination of four different peptide biomarkers selective for the Ara h2 and Ara h3/4 peanut proteins. The performance of this method was compared with that of a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) method with ICP-MS detection of the metal used to tag the antibody for the quantitative peanut protein analysis in food. The limit of detection (LOD) and quantitation of the ICP-MS immunoassay were 2.2 and 5 microg peanuts g(-1) matrix, respectively, the recovery ranged from 86 +/- 18% to 110 +/- 4% and linearity was proved in the 5-50 microg g(-1) range. The LC/MS/MS method allowed us to obtain LODs of 1 and 5 microg protein g(-1) matrix for Ara h3/4 and Ara h2, respectively, thus obtaining significantly higher values with respect to the ELISA ICP-MS method, taking into account the different expression for concentrations. Linearity was established in the 10-200 microg g(-1) range of peanut proteins in the food matrix investigated and good precision (RSD <10%) was demonstrated. Both the two approaches, used for screening or confirmative purposes, showed the power of mass spectrometry when used as a very selective detector in difficult matrices even if some limitations still exist, i.e. matrix suppression in the LC/ESI-MS/MS procedure and the change of the Ag/Ab binding with matrix in the ICP-MS method.     

Particle-packed column versus silica-based monolithic column for liquid chromatography-electrospray-linear ion trap-tandem mass spectrometry multiallergen trace analysis in foods

A bicarbonate buffer-based extraction method for the simultaneous analysis of five nut allergens (Ana o 2, cashew-nut; Cor a 9, hazelnut; Pru 1, almond; Ara h3/4, peanut; Jug r 4, walnut) in cereals and biscuits using liquid chromatography-electrospray-linear ion trap-tandem mass spectrometry (LC-ESI-LIT-MS(2)) was developed and validated. The method was based on our earlier published LC-MS(2)-based method in a research frame aimed at the identification and determination of hidden allergens in foods by using selective biomarker peptides. A C18 particle-packed column and a silica-based C18 monolithic column were compared in terms of chromatographic performances, such as peak shape, resolution, analysis time and selectivity. The C18 particle-packed column exhibited better performances and was further used for method development and validation. By operating under MS(2) selected reaction monitoring (SRM) acquisition mode, linearity, limits of detection (LOD) and quantitation, trueness and precision were evaluated on breakfast samples enriched with a mix of the five nuts. Good linearity of the matrix matched-calibration curves was obtained and detection limit values generally varied from 14 to 55 mg nut/kg matrix. Recoveries were in the 76±4% to 94±3% range with RSD <15%. The capabilities of LIT to perform MS(n) fragmentation was exploited to improve selectivity of the analysis, and the LC-(SRM) MS(2) method was compared in terms of LOD, linearity, precision and accuracy with a LC-(SRM) MS(3) method. Finally, both the LC-MS(2) and LC-MS(3) methods were successfully applied to the analysis of nut traces in commercially available breakfast cereals and biscuits.     

Use of specific peptide biomarkers for quantitative confirmation of hidden allergenic peanut proteins Ara h 2 and Ara h 3/4 for food control by liquid chromatography–tandem mass spectrometry

A liquid chromatography–electrospray-tandem mass spectrometry (LC–ESI-MS–MS) method based on the detection of biomarker peptides from allergenic proteins was devised for confirming and quantifying peanut allergens in foods. Peptides obtained from tryptic digestion of Ara h 2 and Ara h 3/4 proteins were identified and characterized by LC–MS and LC–MS–MS with a quadrupole-time of flight mass analyzer. Four peptides were chosen and investigated as biomarkers taking into account their selectivity, the absence of missed cleavages, the uniform distribution in the Ara h 2 and Ara h 3/4 protein isoforms together with their spectral features under ESI-MS–MS conditions, and good repeatability of LC retention time. Because of the different expression levels, the selection of two different allergenic proteins was proved to be useful in the identification and univocal confirmation of the presence of peanuts in foodstuffs. Using rice crispy and chocolate-based snacks as model food matrix, an LC–MS–MS method with triple quadrupole mass analyzer allowed good detection limits to be obtained for Ara h2 (5 μg protein g⁻¹ matrix) and Ara h3/4 (1 μg protein g⁻¹ matrix). Linearity of the method was established in the 10–200 μg g⁻¹ range of peanut proteins in the food matrix investigated. Method selectivity was demonstrated by analyzing tree nuts (almonds, pecan nuts, hazelnuts, walnuts) and food ingredients such as milk, soy beans, chocolate, cornflakes, and rice crispy. Figure ESI-QTOF-MS mass spectrum of Ara h3/4 triptig digest     

Immuno-mass spectrometry assay of EPI-HNE4, a recombinant protein inhibitor of human elastase

In order to increase the sensitivity of liquid chromatography/mass spectrometry (LC/MS) assays of recombinant proteins for pharmacokinetics studies, we have developed an immuno-mass spectrometry assay for EPI-hNE4, a 6237 Da protein currently developed for respiratory distress syndromes. After immunocapture of the analyte in human plasma with magnetic beads coated with anti-EPI-hNE4 antibodies, the intact protein was eluted and separated in reversed-phase LC and then analysed by tandem mass spectrometry (MS/MS) in selected reaction monitoring (SRM) mode. The problem of analytical interference due to endogenous binding antibodies was addressed by successive steps of acidification and neutralisation before immunocapture. Furthermore, potential variations in the recovery of analyte during sample extraction were compensated for by addition of an internal standard recognised by the antibodies. The precision of the assay remained therefore below 15%. A significant increase in assay sensitivity was achieved since the extraction step allowed sample concentration and removal of matrix components interfering with the electrospray ionisation process. Using 0.4 mL of plasma, a limit of quantification at 0.5 ng/mL (80 pM) was reached, which represents a 10-fold improvement in sensitivity over our previous work using sample precipitation. This technique was able to monitor EPI-hNE4 kinetics in the plasma of human subjects for 36 h after an intravenous administration of 0.125 mg/kg.     

Selective enrichment and quantification of okadaic acid in shellfish using an immunomagnetic‐bead‐based liquid chromatography with tandem mass spectrometry assay

Okadaic acid is a marine biotoxin that primarily occurs in shellfish and can cause diarrheic shellfish poisoning in humans. When analyzing biological samples using liquid chromatography with tandem mass spectrometry, the presence of complex matrices is a major issue. Thus, it is crucial to selectively and simply extract the target analyte from samples and minimize matrix effects simultaneously. To meet this need, an immunomagnetic‐bead‐based liquid chromatography with tandem mass spectrometry method was developed to detect okadaic acid in shellfish. Magnetic beads bound to monoclonal antibody against okadaic acid were used as affinity probes to specifically enrich okadaic acid in samples, which effectively eliminated matrix effects. A magnetic separator was used to aggregate and separate magnetic particles from sample matrices, and methanol was used to elute okadaic acid from the magnetic beads. Standard solution prepared with methanol was employed directly for quantitative analysis. Several experimental conditions were optimized to improve performance. The method is of interest as a rapid (10 min) sample clean‐up and selective enrichment tool, and it showed good linearity and sensitivity, with reported limits of detection and quantitation of 3 and 10 μg/kg, respectively. Fifty‐three shellfish samples from an aquatic products market were tested using this method, and four samples positive for okadaic acid were found.     

超高效液相色谱-四极杆/静电场轨道阱高分辨质谱检测食品中的牛奶过敏原酪蛋白

基于超高效液相色谱-四极杆/静电场轨道阱高分辨质谱系统,建立了食品中牛奶过敏原酪蛋白的快速筛查和定量检测方法。样品经缓冲液提取后,采用5 kD超滤膜去除小分子杂质,得到蛋白质提取液。以数据依赖采集(data-dependent acquisition,DDA)方式获得全扫描质谱图,进行蛋白质定性确证,以平行反应监测(parallel reaction monitoring,PRM)技术对目标特征肽段进行定量分析。针对特征肽段,设计并合成了内标肽和内标物质,以降低基质效应和抵消处理过程中的损失。该方法应用于食品中的α-酪蛋白、β-酪蛋白和κ-酪蛋白的快速筛查和定量检测。结果表明,该方法在5~250μg/L范围内线性关系良好,定量限为0.2~5.5μg/kg,平均回收率在68.8%~104.4%之间,RSD6%。该方法可用于果汁饮料、果酱、面包、早餐谷物中牛奶过敏原酪蛋白的快速筛查和定量分析。     

Liquid chromatography/mass spectrometry compatible approaches for the quantitation of folic acid in fortified juices and cereals using aqueous normal phase conditions

Folic acid was separated under aqueous normal phase (ANP) conditions with Diamond Hydride™ columns and quantitated in fortified cereal and juice matrices using high performance liquid chromatography/ultraviolet absorption (HPLC-UV) and liquid chromatography/mass spectrometry (LC–MS) based methodologies. The folic acid was well-resolved from matrix components under the ANP conditions studied and allowed for the direct analyses of the fortified juices and cereals without the sample cleanup that is often required for other reported LC-based approaches. The calibration curve obtained from the LC–MS analyses demonstrated good linearity (R² = 0.9997) in the studied concentration range of 0.05–0.5 mg/L. The spiked flour percent recovery was 90% with HPLC-UV and 91% with LC–MS. Spiked juice percent recovery was 102% with LC–MS. However, analyses of juices were unsatisfactory either in terms of recovery or sensitivity. Therefore, analyses of juices will either need to be performed by LC–MS or, if by HPLC-UV, will require sample cleanup. Three cereals and one juice were analyzed with the methods.     

固相萃取/超高效液相色谱-质谱法筛查及检测养鱼河水中抗生素

建立了固相萃取/超高效液相色谱-串联三重四极杆质谱(UPLC-MS/MS)快速检测养鱼河水中4类(喹诺酮类、氯霉素类、四环素类以及磺胺类)15种抗生素的分析方法。取500 m L水样过滤,用盐酸调至p H4.0,加入0.5 g乙二胺四乙酸二钠(Na2EDTA)混匀后,再用HLB固相萃取柱对水样进行富集。利用超高效液相色谱-串联四极杆飞行时间质谱(UPLC-Q-TOF MS)结合基于UNIFI软件的常用抗生素数据库对养鱼河水中可能存在的抗生素进行快速筛查,筛查结果主要为喹诺酮类、氯霉素类、四环素类、磺胺类4类15种抗生素。为了提高检测结果的准确性,使用UPLC-MS/MS采用分时段多反应监测离子模式(MRM)分析样品中的抗生素,并用外标法定量。15种抗生素的空白基质加标回收率为61.0%~98.4%,相对标准偏差(n=3)为4.6%~14.0%,其线性关系良好,相关系数r≥0.990,检出限为0.01~0.3 ng/L。该方法灵敏度较高,重复性好,可用于对北京养鱼河水中抗生素的检测。     

Liquid-phase microextraction for sample preparation in analysis of unconjugated anabolic steroids in urine

The applicability of in-vial two-phase liquid-phase microextraction (LPME) in porous hollow polypropylene fiber was studied for the sample preparation of unconjugated anabolic steroids in urine. Four different anabolic steroids - metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol - were used as test compounds and methyltestosterone as an internal standard. A standard two-phase LPME method for use with liquid chromatography/mass spectrometry (LC/MS) was set up and the influence of different parameters, including the nature of organic solvent, extraction time, salting-out and temperature, on the LPME process was investigated. Taking advantage of the preliminary studies, a novel two-phase LPME method utilizing simultaneous in-fiber silylation was developed and validated for gas chromatographic/mass spectrometric (GC/MS) analysis of a danazol metabolite in urine. In all, LPME allowed a very straightforward, simple and selective way to prepare urine samples for steroid analysis, being most suitable for hydrophobic steroids. The LPME method with in-fiber derivatization for GC/MS analysis exhibited high sensitivity, repeatability and linearity and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine matrix without any other steps in sample pretreatment.     

The highly selective capture of phosphopeptides by zirconium phosphonate-modified magnetic nanoparticles for phosphoproteome analysis

The highly selective capture of phosphopeptides from proteolytic digests is a great challenge for the identification of phosphoproteins by mass spectrometry. In this work, the zirconium phosphonate-modified magnetic Fe₃O₄/SiO₂ core/shell nanoparticles have been synthesized and successfully applied for the selective capture of phosphopeptides from complex tryptic digests of proteins before the analysis of MALDI-TOF mass spectrometry with the desired convenience of sample handling. The ratio of magnetic nanoparticle to protein and the incubation time for capturing phosphopeptides from complex proteolytic digests were investigated, and the optimized nanoparticle-to-protein ratio and incubation time were between 15:1 to 30:1 and 30 min, respectively. The excellent detection limit of 0.5 fmol β-casein has been achieved by MALDI-TOF mass spectrometry with the specific capture of zirconium phosphonate-modified magnetic Fe₃O₄ nanoparticles. The great specificity of zirconium phosphonate-modified magnetic Fe₃O₄ nanoparticles to phosphopeptides was demonstrated by the selective capture of phosphopeptides from a complex tryptic digest of the mixture of α-casein and bovine serum albumin at molar ratio of 1 to 100 in MALDI-TOF-MS analysis. An application of the magnetic nanoparticles to selective capture phosphopeptides from a tryptic digest of mouse liver lysate was further carried out by combining with nano-LC-MS/MS and MS/MS/MS analyses, and a total of 194 unique phosphopeptides were successfully identified.     

Full-scan accurate mass selectivity of ultra-performance liquid chromatography combined with time-of-flight and orbitrap mass spectrometry in hormone and veterinary drug residue analysis

The applicability of ultra-performance liquid chromatography (UPLC) combined with full-scan accurate mass time-of-flight (TOF) and Orbitrap mass spectrometry (MS) to the analysis of hormone and veterinary drug residues was evaluated. Extracts from blank bovine hair were fortified with 14 steroid esters. UPLC-Orbitrap MS performed at a resolving power of 60,000 (FWHM) enabled the detection and accurate mass measurement (<3 ppm error) of all 14 steroid esters at low ng/g concentration level, despite the complex matrix background. A 5 ppm mass tolerance window proved to be essential to generate highly selective reconstructed ion chromatograms (RICs) having reduced background from the hair matrix. UPLC-Orbitrap MS at a lower resolving power of 7500 and UPLC-TOFMS at mass resolving power 10,000 failed both to detect all of the steroid esters in hair extracts owing to the inability to mass resolve analyte ions from co-eluting isobaric matrix compounds. In a second application, animal feed extracts were fortified with coccidiostats drugs at levels ranging from 240 to 1900 ng/g. UPLC-Orbitrap MS conducted at a resolving power of 7500 and 60,000 and UPLC-TOFMS detected all of the analytes at the lowest investigated level. Thanks to the higher analyte-to-matrix background ratio, the utilization of very narrow mass tolerance windows in the RIC was not required. This study demonstrates that even when the targeted sample preparation from conventional LC-MS/MS is applied to UPLC with full-scan accurate mass MS, false compliant (false negative) results can be obtained when the mass resolving power of the MS is insufficient to separate analyte ions from isobaric co-eluting sample matrix ions. The current trend towards more generic and less selective sample preparation is expected to aggravate this issue further.     

Optimal preparation methods for automated matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry profiling of low molecular weight proteins and peptides

Mass spectrometry (MS) profiling of the proteome and peptidome for disease‐associated patterns is a new concept in clinical diagnostics. The technique, however, is highly sensitive to external sources of variation leading to potentially unacceptable numbers of false positive and false negative results. Before MS profiling can be confidently implemented in a medical setting, standard experimental methods must be developed that minimize technical variance. Past studies of variance have focused largely on pre‐analytical variation (i.e., sample collection, handling, etc.). Here, we examined how factors at the analytical stage including the matrix and solid‐phase extraction influence MS profiling. Firstly, a standard peptide/protein sample was measured automatically by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS across five consecutive days using two different preparation methods, dried droplet and sample/matrix, of four types of matrix: α‐cyano‐4‐hydroxycinnamic acid (HCCA), sinapinic acid (SA), 2,5‐dihydroxybenzoic acid (DHB) and 2,5‐dihydroxyacetophenone (DHAP). The results indicated that the matrix preparation greatly influenced a number of key parameters of the spectra including repeatability (within‐day variability), reproducibility (inter‐day variability), resolution, signal strength, background intensity and detectability. Secondly, an investigation into the variance associated with C8 magnetic bead extraction of the standard sample prior to automated MS profiling demonstrated that the process did not adversely affect these same parameters. In fact, the spectra were generally more robust following extraction. Thirdly, the best performing matrix preparations were evaluated using C8 magnetic bead extracted human plasma. We conclude that the DHAP prepared according to the dried‐droplet method is the most appropriate matrix to use when performing automated MS profiling. Copyright © 2009 John Wiley & Sons, Ltd.     

粮谷中双苯唑菌醇残留量的液相色谱-质谱/质谱检测

建立了糙米、玉米、大麦、小麦、荞麦、大米、高粱、燕麦中双苯唑菌醇残留量的液相色谱-质谱/质谱测定方法.样品经乙腈提取,氨基(NH2)固相萃取柱净化,液相色谱-质谱/质谱仪测定,外标法定量.方法的定量下限为0.025 mg/kg,粮谷样品在0.025、0.050、0.500 mg/kg添加水平的平均回收率为 79% ～92%,相对标准偏差为4.8% ～10.8%,可以满足进出口谷物中双苯唑菌醇残留量的检测需要.     

Laserspray ionization using an atmospheric solids analysis probe for sample introduction

A newly introduced high sensitivity laserspray (LSI) mass spectrometry (MS) method that uses laser ablation of a matrix/analyte mixture at atmospheric pressure (AP) to obtain multiply charged ions from nonvolatile as well as high-mass compounds is now implemented using a simple probe device. The probe used in the LSI approach was originally designed for sample introduction into an AP ionization source using the atmospheric solids analysis probe (ASAP) method. Multiply charged mass spectra of peptides and proteins in 2,5-dihydroxybenzoic acid matrix were readily obtained on two mass spectrometers from different manufacturers with sample introduction using melting point tubes. Here we demonstrate rapid analysis by placing four peptide and protein samples on a single melting point tube. Mass spectra were obtained at high-resolution and using ion mobility spectrometry/MS.     

Analysis of amprolium by hydrophilic interaction liquid chromatography–tandem mass spectrometry

We present a fast liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the analysis of the coccidiostat amprolium in food samples. Tandem mass spectrometry in a triple quadrupole was used for quantitative purposes, and the information from multiple-stage mass spectrometry in an ion-trap mass analyzer contributed to fragmentation studies. Hydrophilic interaction liquid chromatography (HILIC) in a Fused-Core™ column using isocratic elution (acetonitrile:formic acid/ammonium formate buffer pH 4, 50 mM (60:40)) successfully analyzed this compound in less than 3 min. The HILIC system was coupled to heated electrospray-MS/MS using highly selective-selected reaction monitoring (H-SRM) to improve sensitivity and selectivity for the analysis of amprolium, after a simple sample treatment based on an “extract and shoot” strategy. Accurate mass measurements were performed to identify the interfering compound responsible for causing matrix ion enhancement in the signal of amprolium. The addition of l-carnitine (the interfering compound) (1 μg L⁻¹) to standards and sample extracts allowed the use of the external calibration method for quantitative purposes. The LC–MS/MS (H-SRM) method showed good precision (relative standard deviation, RSD, lower than 13%), accuracy and linearity and allowed the determination of amprolium down to the ppb level (LODs between 0.1 and 0.6 μg kg⁻¹).     

Development and validation of a method for the quantification of milk proteins in food products based on liquid chromatography with mass spectrometric detection

The protection of allergic consumers is crucial to the food industry. Therefore, accurate methods for the detection of food allergens are required. Targeted detection of selected molecules by MS combines high selectivity with accurate quantification. A confirmatory method based on LC/selected reaction monitoring (SRM)-MS/MS was established and validated for the quantification of milk traces in food. Tryptic peptides of the major milk proteins beta-lactoglobulin, beta-casein, alphaS2-casein, and K-casein were selected as quantitative markers. Precise quantification was achieved using internal standard peptides containing isotopically labeled amino acids. For each peptide, qualifier and quantifier fragments were selected according to Commission Decision 2002/657/EC. A simple sample preparation method was established without immunoaffinity or SPE enrichment steps for food matrixes containing different amounts of protein, such as baby food, breakfast cereals, infant formula, and cereals. Intermediate reproducibility, repeatability, accuracy, and measurement uncertainty were determined for each matrix. LOD values of 0.2-0.5 mg/kg, e.g., for beta-lactoglobulin, were comparable to those obtained with ELISA kits. An LOQ of approximately 5 mg/kg, expressed as mass fraction skim milk powder, was validated in protein-rich infant cereals. The obtained validation data show that the described LC/SRM-MS/MS approach can serve as a confirmatory method for the determination of milk traces in selected food matrixes.     

Analysis of acrylamide in different foodstuffs using liquid chromatography–tandem mass spectrometry and gas chromatography–tandem mass spectrometry

Acrylamide levels over a wide range of different food products were analysed using both liquid chromatography–tandem mass spectrometry (HPLC–MS–MS) and gas chromatography–tandem mass spectrometry (GC–MS–MS). Two different sample preparation methods for HPLC–MS–MS analysis were developed and optimised with respect to a high sample throughput on the one hand, and a robust and reliable analysis of difficult matrices on the other hand. The first method is applicable to various foods like potato chips, French fries, cereals, bread, and roasted coffee, allowing the analysis of up to 60 samples per technician and day. The second preparation method is not as simple and fast but enables analysis of difficult matrices like cacao, soluble coffee, molasses, or malt. In addition, this method produces extracts which are also well suited for GC–MS–MS analysis. GC–MS–MS has proven to be a sensitive and selective method offering two transitions for acrylamide even at low levels up to 1 μg kg⁻¹. For the respective methods the repeatability (n=10), given as coefficient of variation, ranged from 3% (acrylamide content of 550 μg kg⁻¹) to 12% (acrylamide content of 8 μg kg⁻¹) depending on the food matrix. The repeatability (n=3) for different food samples spiked with acrylamide (5–1500 μg kg⁻¹) ranged from 1 to 20% depending on the spiking level and the food matrix. The limit of quantification (referred to a signal-to-noise ratio of 9:1) was 30 μg kg⁻¹ for HPLC–MS–MS and 5 μg kg⁻¹ for GC–MS–MS. It could be demonstrated that measurement uncertainties were not only a result of analytical variability but also of inhomogeneity and stability of the acrylamide in food.     

Determination of two oxy-pyrimidine metabolites of diazinon in urine by gas chromatography/mass selective detection and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry

An analytical method was developed for the determination in urine of 2 metabolites of diazinon: 6-methyl-2-(1-methylethyl)-4(1H)-pyrimidinone (G-27550) and 2-(1-hydroxy-1-methylethyl)-6-methyl-4(1H)-pyrimidinone (GS-31144). Two of the urine sample preparation procedures presented rely on gas chromatography/mass selective detection (GC/MSD) in the selected ion monitoring mode for determination of G-27550. For fast sample preparation and a limit of quantitation (LOQ) of 1.0 ppb, urine samples were purified by using ENV+ solid-phase extraction (SPE) columns. For analyte confirmation at an LOQ of 0.50 ppb, classical liquid/liquid partitioning was used before further purification in a silica SPE column. An SPE sample preparation procedure and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry (LC/ESI/MS/MS) were used for both G-27550 and GS-31144. The limit of detection was 0.01 ng for G-27550 with GC/MSD, and 0.016 ng when LC/ESI/MS/MS was used for both G-27550 and GS-31144. The LOQ was 0.50 ppb for G-27550 when GC/MSD and the partitioning/SPE sample preparation procedure were used, and 1.0 ppb for the SPE only sample preparation procedure. The LOQ was 1.0 ppb for both analytes when LC/ESI/MS/MS was used.     

Towards clinical proteomics analysis

To understand accurate protein dynamics, a highly reproducible proteomics analytical method is required. The acquired thus knowledge will lead to the diagnosis, treatment and protection against diseases. This review deals with proteomics analysis from a view of sample pre-treatment, sensitivity and reproducibility of the methods, including two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), liquid chromatography-tandem mass spectrometry (LC-MS/MS). and fluorogenic derivatization (FD)-LC-MS/MS.     

A high-throughput monoamine oxidase inhibition assay using liquid chromatography with tandem mass spectrometry

A highly efficient method utilizing liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed and employed for high-throughput screening of compounds for monoamine oxidase (MAO) inhibition. The method used kynuramine as a common substrate for both MAO-A and MAO-B in incubations, and the 4-hydroxyquinoline (4-HQ) resulting from deamination of kynuramine followed by intramolecular condensation was analyzed using LC/MS/MS; formation of 4-HQ was used as the marker of MAO activity to evaluate the effects of test compounds. Isocratic liquid chromatography was applied to reduce the run time to 2 min. Because of the high specificity and sensitivity of detection of 4-HQ by LC/MS/MS, this method was able to use MAO enzymes at very low concentrations and to perform short incubations; as a result, consumable cost was minimized, and sample preparations were completely avoided. The inhibition data are highly reproducible, and the IC(50) values determined by the method are in good agreement with literature values. The results suggest that this method is very robust and can be used as a generic approach to screen for MAO inhibitors in drug discovery.