Strategic use of immunoprecipitation and LC/MS/MS for trace-level protein quantification: myosin light chain 1, a biomarker of cardiac necrosis

Myosin light chain 1 (Myl3) is a 23-kDa isoform of one of the subunits of myosin, a protein involved in muscle contraction. Myl3 is presently being studied as a biomarker of cardiac necrosis to predict drug-induced cardiotoxicity, and in the work presented here, an LC/MS/MS assay was developed and validated to measure Myl3 in rat serum. The key steps in this approach involved immunoaffinity purification of Myl3 from serum followed by on-bead digestion with trypsin to release a surrogate peptide. This tryptic peptide was quantified using a synthetic peptide standard and a corresponding stable isotope-labeled internal standard, and the results were stoichiometrically converted to Myl3 serum concentrations. Myl3 concentrations were corrected for peptide recovery following immunoprecipitation and digestion (85%) and showed excellent agreement with synthetic peptide standards. Both the synthetic peptide and His-Myl3 protein were used to evaluate assay accuracy (% RE) and precision (% CV), which were measured on each of 3 days. The synthetic peptide was evaluated over the range of 0.073-7.16 nM, while Myl3 protein QC samples prepared in rat serum were evaluated over the range of 0.13-6.62 nM. To prepare control matrix, endogenous Myl3 was immunodepleted from pooled rat serum. Peptide interday accuracy and precision did not exceed 7.6 and 11.1%, and Myl3 interday accuracy and precision did not exceed 12.9 and 13.2%, respectively. Data are presented from the application of this assay to establish a time course in which rats demonstrated a marked increase in Myl3 serum concentrations following administration of isoproterenol, a beta-adrenergic receptor agonist known to induce cardiac injury. This assay is an example of a larger effort in our laboratory to use LC/MS/MS in conjunction with immunoaffinity techniques to evaluate candidate biomarkers of target organ toxicity and to expedite the development of biomarker assays for drug development.     

Quantification and rapid metabolite identification in drug discovery using API time-of-flight LC/MS

Liquid chromatography/mass spectrometry (LC/MS), utilizing a time-of-flight (TOF) mass analyzer, has been evaluated and applied to problems in bioanalysis for pharmacokinetics and drug metabolism. The data obtained by TOF MS differ from those obtained using quadrupole mass spectrometer instruments in that full-scan spectra can be routinely collected with greater sensitivity and speed. Both quantitative and qualitative information, including compound concentration in rat plasma and full-scan atmospheric pressure ionization mass spectra, are concurrently obtained. This approach has been used to characterize the disposition of several drug compounds that have been simultaneously dosed to rats in a cassette format. Quantitation limits in the 5-25 ng/mL range (approximately 20 nM) were obtained from nominal mass chromatograms (0.5 Da resolution). A reference lock mass was used to provide accurate mass measurement to reach third decimal place accuracy in the monoisotopic molecular weight. An improvement in quantitation limits was demonstrated after using accurate mass determinations. Several possible preliminary drug metabolites were confirmed or refuted, based on accurate mass. The trend of metabolite formation and clearance was qualitatively evaluated.     

Analysis of phenothiazine and its derivatives using LC/electrochemistry/MS and LC/electrochemistry/fluorescence

The on-line electrochemical conversion of phenothiazine and its derivatives after liquid chromatographic separation has been studied by mass spectrometry and fluorescence spectroscopy. In an electrochemical cell consisting of porous glassy carbon, the phenothiazines are readily converted to oxidized products, which can be detected by on-line fluorescence spectroscopy and mass spectrometry. The method allows rapid investigations on the electrochemical oxidation pathways, as demonstrated for phenothiazine itself. The phenothiazine derivatives are transferred into their strongly fluorescent sulfoxides. Based on this reaction, an LC/electrochemistry/fluorescence method was developed that allows for limits of detection between 5 x 10(-9) and 4 x 10(-8) mol/L and limits of quantification between 2 x 10(-8) and 1 x 10(-7) mol/L for the individual phenothiazines. The linear ranges comprised three decades starting at the limit of quantification.     

Extraction and quantification of phytoestrogens in foods using automated solid-phase extraction and LC/MS/MS

Phytoestrogens are a group of polyphenolic plant metabolites that can induce biological responses. Their bioactivity is based on their similarity to 17beta-estradiol and their ability to bind to the beta-estrogen receptor. Although epidemiological data are inconclusive, phytoestrogens are considered to be beneficial for a variety of conditions, for example, hormone-related cancers like breast and prostate cancer. To investigate the biological effects of these compounds and to assess the exposure of larger cohorts or the general public, reliable data on the phytoestrogen content of food is necessary. Previously, food analysis for phytoestrogens was performed using either HPLC-UV or GC/MS. Here, we describe the development of the first generic method for the analysis of phytoestrogens in food, using automated solid-phase extraction and liquid chromatography-tandem mass spectrometry. The presented method shows a good reproducibility and can be easily adapted to other phytoestrogens if required.     

Quantification of o,o'-dityrosine,o-nitrotyrosine,and o-tyrosine in cat urine samples by LC/ electrospray ionization-MS/MS using isotope dilution

Quantification of o-tyrosine, o-nitrotyrosine, and o,o'dityrosine from cat urine samples was achieved by LC/ electrospray ionization-MS/MS (LC/ESI-MS/MS) using an isotope dilution technique in multiple reaction monitoring mode before butylation of o,o'-dityrosine and after butylation of o-tyrosine and o-nitrotyrosine. This novel approach of amino acids butylation enhanced the MS response by a factor of 7-fold for o-tyrosine and 6-fold for o-nitrotyrosine and decreased the overall chemical background noise. Butylation of o,o'-dityrosine resulted in a lower MS response as a result of the formation of both mono- and doubly butylated species. The mean recovery for the oxidized amino acids was estimated at 73 +/- 2%. The limits of quantitation of NO2-Tyr butyl ester, o-Tyr butyl ester, and di-Tyr in cat urine samples were calculated at 14.5, 28.2 and 140.9 nM, respectively. The oxidized amino acids levels in cat urine extracts ranged from 157 to 250 ng/day for o-Tyr and from 3,289 to 11,803 ng/day for di-Tyr. NO2-Tyr was found in only two urine extracts at levels below 58 ng/day. A certain trend of correlation was observed between o,o'-dityrosine and o-tyrosine when comparing these values against their respective creatinine amounts. A comparison of the data gathered from the ThermoFinnigan TSQ 7000 and Micromass Q-TOF instruments revealed several advantages of using the Q-TOF regarding the exact mass measurement, a lower ion suppression effect and the possibility to perform analyses in full scan product ion mode. These results demonstrate that a Q-TOF instrument can be a good alternative to classical triple quadrupole for quantitative purposes on a relatively small linear dynamic range (4 orders of magnitude for the Q-TOF, as compared to 6 for the triple quadrupole).     

Combination of LC/TOF-MS and LC/Ion trap MS/MS for the identification of diphenhydramine in sediment samples

Diphenhydramine (Benadryl) is a popular over-the-counter antihistaminic medication used for the treatment of allergies. After consumption, excretion, and subsequent discharge from wastewater treatment plants, it is possible that diphenhydramine will be found in environmental sediments due to its hydrophobicity (log P = 3.27). This work describes a methodology for the first unequivocal determination of diphenhydramine bound to environmental sediments. The drug is removed from the sediments by accelerated solvent extraction and then analyzed by liquid chromatography with a time-of-flight mass spectrometer and an ion trap mass spectrometer. This combination of techniques provided unequivocal identification and confirmation of diphenhydramine in two sediment samples. The accurate mass measurements of the protonated molecules were m/z 256.1703 and 256.1696 compared to the calculated mass of m/z 256.1701, resulting in errors of 0.8 and 2.3 ppm. This mass accuracy was sufficient to verify the elemental composition of diphenhydramine in each sample. Furthermore, accurate mass measurements of the primary fragment ion were obtained. This work is the first application of time-of-flight mass spectrometry for the identification of diphenhydramine and shows the accumulation of an over-the-counter medication in aquatic sediments at five different locations.     

GlycoFish: a database of zebrafish N-linked glycoproteins identified using SPEG method coupled with LC/MS

Zebrafish (Danio rerio) is a model organism that is used to study the mechanisms and pathways of human disorders. Many dysfunctions in neurological, development, and neuromuscular systems are due to glycosylation deficiencies, but the glycoproteins involved in zebrafish embryonic development have not been established. In this study, a mass spectrometry-based glycoproteomic characterization of zebrafish embryos was performed to identify the N-linked glycoproteins and N-linked glycosylation sites. To increase the number of glycopeptides, proteins from zebrafish were digested with two different proteases--chymotrypsin and trypsin--into peptides of different length. The N-glycosylated peptides of zebrafish were then captured by the solid-phase extraction of N-linked glycopeptides (SPEG) method and the peptides were identified with an LTQ OrbiTrap Velos mass spectrometer. From 265 unique glycopeptides, including 269 consensus NXT/S glycosites, we identified 169 different N-glycosylated proteins. The identified glycoproteins were highly abundant in proteins belonging to the transporter, cell adhesion, and ion channel/ion binding categories, which are important to embryonic, organ, and central nervous system development. This proteomics data will expand our knowledge about glycoproteins in zebrafish and may be used to elucidate the role that glycosylation plays in cellular processes and disease. The glycoprotein data are available through the GlycoFish database (http://betenbaugh.jhu.edu/GlycoFish) introduced in this paper.     

Analysis of perchlorate in water and soil by electrospray LC/MS/MS

A method has been developed for the determination of perchlorate in water and soil matrixes using electrospray liquid chromatography/mass spectrometry/mass spectrometry. Perchlorate is quantitated by monitoring the ion signal from mass 83, which is formed by a loss of an oxygen atom from the perchlorate molecular ion. The method was developed to be effective and economical in production laboratory analysis of perchlorate in environmental water and soil samples. Data were gathered to define method sensitivity, performance, selectivity, and robustness. Analyte stability, method susceptibility to interferences, and the reliability of the chlorine isotope ratio as an identification tool were examined. The aqueous method detection limit (MDL) is 0.05 microg/L and was determined using an actual groundwater matrix. The soil MDL is 0.5 microg/kg and was determined using Ottawa sand. The stability study was performed by spiking water samples at 0.25, 10, and 20 microg/L and analyzing them 50 days later. Acceptable recoveries were obtained for all samples. The relative standard deviation (RSD) for the replicate analyses in the stability study indicates that the method is capable of RSD values less than 5% in a relatively clean groundwater matrix. The ionization suppression study was performed by spiking water samples containing 1000 mg/L carbonate, chloride, and sulfate with 0.05 and 0.5 microg/L perchlorate and then measuring the recovery of the spike. The results indicate that the procedure does not have significant suppression effects at the high salt levels tested. Calibration, quality control sample, field sample, and suppression study data were combined to examine isotope ratio reliability. The results of that work show that chlorine isotope ratios can be used to define statistical process control limits for use as an additional analyte identification tool.     

Evaluation of triterpene glycoside estrogenic activity using LC/MS and immunoaffinity extraction

We present a study on the mass spectral as well as the binding properties of three triterpene glycosides (cimicifugoside, cimiracemoside F, 27-deoxyactein) contained in black cohosh to the ligand binding domain of estrogen receptor beta (ER-beta). Using affinity ultrafiltration and LC/ MS detection, initial experiments using estradiol and the phytoestrogens daidzein and genistein (compounds known to bind ER-beta) were performed to serve as positive controls. The same affinity techniques and LC/MS procedures were then employed to show that neither the triterpene glycosides nor their enzymatically prepared aglycons bound significantly to ER-beta, except for 27-deoxyactein aglycon, which showed weak binding affinity (4%). Additionally, metabolites of the aglycons were prepared by incubation with female human liver microsomes and subjected to binding experiments with ER-beta. No significant binding of the metabolites to the receptor was observed. Further studies are needed to fully characterize whether these triterpene glycosides as well as other components of black cohosh in this plant extract bind to the estrogen receptor alpha (ER-alpha).     

Development and validation of an LC/MS/MS procedure for the quantification of endogenous myo-inositol concentrations in rat brain tissue homogenates

myo-Inositol is being investigated as a biomarker to monitor disease states involving the central nervous system. We have developed and validated a quantitative method to study endogenous myo-inositol metabolism in rat brain tissue. Tissue samples were homogenized, and their myo-inositol content was determined using spiked calibration curves and mass spectrometry. The assay was validated on an LC/MS/MS platform, and specificity was evaluated using accurate mass measurements. A novel chiral LC/MS/MS method was also developed to resolve myo-inositol from other endogenous inositol epimers and confirm the selectivity of the quantitative procedure. The validated method is selective, convenient, precise (<15% RSD), accurate (<15% RE), and sensitive over a linear range of 0.100-100 microg/mL. This method could potentially be used as an instrument for monitoring pathological conditions related to psychotherapeutics, as well as a tool for screening curative pharmaceuticals for efficacy.     

Characterization of dyes and other pollutants in the effluent of a textile company by LC/NMR and LC/MS

LC/NMR and LC/MS (the latter technique in the MSn mode) were used to characterize the organic constituents of industrial wastewater with emphasis on polar, nonvolatile compounds. In the effluent of a textile company, various compounds such as anthraquinone-type dyes and their byproducts, a fluorescent brightener, a byproduct from polyester production, and auxiliaries such as anionic and nonionic surfactants and their degradation products were identified. It is shown that the combined use of both hyphenated techniques provides complementary structural information. If applied under comparable chromatographic conditions, they are well-suited for the nontarget analysis.     

水产品中微囊藻毒素ELISA和LC/MS/MS检测

建立了水产品中微囊藻毒素ELISA和LC/MS/MS测定方法,样品经过提取、净化后,测定结果显示:ELISA最低检测限量为1.00μg/kg,1.00、5.00和10.0μg/kg 3个浓度添加水平,测定回收率范围为75.7%-98.7%,平均回收率为88.0%,RSD范围为5.52%-9.21%;LC/MS/MS最低检测限量为0.3μg/kg,5.00、15.0和20.0μg/kg 3个浓度添加水平,测定回收率范围为86.0%-109%,平均回收率99.1%,RSD范围0.65%-2.37%。两种方法20份实际样品测定结果显示:阴性样品和阳性样品符合率为100%,共有5份样品中含有MC-LR,当地监管部门应引起足够的重视,定时开展监测和信息公布。     

Development of a method for quantification of acrolein-deoxyguanosine adducts in DNA using isotope dilution-capillary LC/MS/MS and its application to human brain tissue

Acrolein is a highly reactive alpha,beta-unsaturated aldehyde and is known to react with DNA forming exocyclic acrolein-deoxyguanosine adducts (Acro-dG). These aldehyde-DNA lesions may play a role in mutagenesis, carcinogenesis, and neurodegenerative diseases. In the present work, we described the development and evaluation of a highly sensitive and selective capillary liquid chromatography nanoelectrospray isotope dilution tandem mass spectrometry method for quantitatively analyzing Acro-dG in DNA hydrolysates. This was achieved by applying a stable isotope-labeled analogue Acro-dG-13C10,15N5 as an internal standard to the DNA to be analyzed and then hydrolyzing the DNA enzymatically to nucleosides. The acrolein-modified nucleosides were separated from normal nucleosides by capillary liquid chromatography and quantified by a high-capacity ion trap mass spectrometer in the MS/MS mode. The developed method achieved attomole-level sensitivity (limit of detection was 10 fg, 31 amol on column) for detection of pure Acro-dG adduct standards. The limit of quantification of Acro-dG adducts obtained in 10 mug of DNA hydrolysates was 1.5 fmol, which corresponded to 50 adducts/10(9) normal nucleosides. Application of this method to the analysis of Acro-dG adducts in acrolein (10-fold)-treated calf thymus DNA showed approximately 830 lesion/10(6) DNA nucleosides using as low as 50 ng of DNA. Application of this method to DNA samples (1-2 mug) isolated from brain tissues from Alzheimer's disease subjects and age-matched controls demonstrated 2800-5100 Acro-dG adducts/10(9) DNA nucleosides. Statistically significant differences (P < 0.05) in levels of Acro-dG between AD subjects and controls were observed in DNA isolated from the hippocampus/parahippocampal gyrus.     

Development of LC/MS/MS methods for cocktail dosed Caco-2 samples using atmospheric pressure photoionization and electrospray ionization

Good reliability of Caco-2 permeability studies requires competent sampling and analytical methods to ensure the comparability of day-to-day experiments. In this work, two n-in-one LC/MS/MS methods based on two different ionization techniques were developed and validated for a group of reference compounds; eight of them are recommended by the Food and Drug Administration (FDA) for the evaluation of oral drug permeability. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), as an interface for quantitative LC/MS analysis was evaluated in comparison to the electrospray ionization (ESI). Generally, the validation parameters, including sensitivity, accuracy, and repeatability, were comparable for the APPI and ESI methods. The main difference was that the linear quantitative range of APPI was 3-4 orders of magnitude (r(2) >/= 0.998) whereas in ESI it was typically 2-3 orders of magnitude (r(2) >/= 0.990). By the APPI and ESI methods, the simultaneous analysis of nine highly heterogeneous compounds was achieved within 5.5-7 min, which leads to significant savings in time and cost of the analyses. The successful validation data indicate the usefulness of both the methods for the rapid and sensitive (LOD values typically 相似文献   

Analysis of perchlorate in water by reversed-phase LC/ESI-MS/MS using an internal standard technique

A new method was developed for the analysis of perchlorate in water by using reversed-phase liquid chromatograhy/electrospray ionization-mass spectrometry/mass spectrometry (LC/ESI-MS/MS) in the negative ESI mode. Selective and sensitive perchlorate detection was obtained by monitoring the 35ClO4- --> 35ClO3- and 37ClO4- --> 37ClO3- mass transitions. The 35ClO4- --> 35ClO3- transition was quantitated against the internal standard oxygen-labeled sodium perchlorate (NaCl18O4). Sample pretreatment for the removal of major common anions and dissolved metal ions along with internal standard quantitation sufficiently compensated for ion suppression caused by the matrix. The 37ClO4- --> 37ClO3- transition was examined to provide additional specificity. The method sensitivity, accuracy, and precision were investigated by analyzing fortified blank samples, field samples, and performance evaluation samples. The results (1.01-13.5 microg/L) for the proficiency evaluation samples differed from the certified values (1.04-14.1 microg/L) by 3-18%. The developed reversed-phase LC/ESI-MS/MS method was rapid, accurate, and reproducible. The calculated method detection limits were 0.007 microg/L for deionized reagent water and 0.009 microg/L for synthesized reagent water, respectively. The minimum reporting limit was conservatively set to 0.05 microg/L.     

Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards

Methods for the absolute quantification of a membrane protein are described using isotopically labeled or unlabeled synthetic peptides as standards. Synthetic peptides are designed to mimic peptides that are cleaved from target analyte proteins by proteolytic or chemical digestion, and the peptides selected serve as standards for quantification by LC/MS/MS on a triple quadrupole mass spectrometer. The technique is complementary to relative quantification techniques in widespread use by providing absolute quantitation of selected targets with greater sensitivity, dynamic range, and precision. Proteins that are found to be of interest by global proteome searches can be selected as targets for quantitation by the present method. This method has a much shorter analytical cycle time (minutes versus hours for the global proteome experiments), making it well suited for high-throughput environments. The present approach using synthetic peptides as standards, in conjunction with proteolytic or chemical cleavage of target proteins, allows mass spectrometry to be used as a highly selective detector for providing absolute quantification of proteins for which no standards are available. We demonstrate that quantification is simple and reliable for the integral membrane protein rhodopsin with reasonable recoveries for replicate experiments using low-micromolar solutions of rhodopsin from rod outer segments.     

Multiresidue analysis of multiclass pesticides in lavandin essential oil by LC/MS/MS using the scheduled selected reaction monitoring mode

In this paper we describe the development of the first multiclass pesticide residue method applied to essential oils. A total of 70 pesticides covering a wide range of polarity and currently used on essential oil crops have been included in the method. The procedure consists of a 10-fold dilution of lavandin essential oil followed by a direct injection analysis by liquid chromatography/tandem mass spectrometry. The system used is an API 4000 QTrap equipped with an electrospray ionization interface and operating in scheduled selected reaction monitoring acquisition mode. Matrix effects were evaluated by comparing the slopes of matrix-matched and solvent-based calibration curves. Weak signal suppression or enhancement (<20%) was observed for most of the compounds. Method sensitivity was determined statistically by the injection of five matrix-matched calibration curves with the distribution's normality and the variance's homogeneity checked before establishment of a suitable regression model. Limits of detection (LODs) and quantification (LOQs) were then determined using the blank standard's deviation and the slope of the mean curve. The analytical method has been validated for 67 of the 70 pesticides and meets the following LOQs: ≤1 μg/L for 9 pesticides, ≤5 μg/L for 44, ≤10 μg/L for 9, and ≤20 μg/L for 5.     

Comparison of LC/MS and SFC/MS for screening of a large and diverse library of pharmaceutically relevant compounds

The search for greater speed of analysis has fueled many innovations in high-performance liquid chromatography (HPLC), such as the use of higher pressures and smaller stationary-phase particles, and the development of monolithic columns. Alternatively, one might alter the chromatographic mobile phase. The low viscosity and high diffusivity of the mobile phase in supercritical fluid chromatography (SFC) allows higher flow rates and lower pressure drops than is possible in traditional HPLC. In addition, SFC requires less organic, or aqueous-organic, solvent than LC (important in preparative-scale chromatography) and provides an alternative, normal-phase retention mechanism. But fluids that are commonly used as the main mobile-phase component in SFC, such as CO2, are relatively nonpolar. As a result, SFC is commonly believed to only be applicable to nonpolar and relatively low-polarity compounds. Here we build upon recent work with SFC of polar and ionic compounds and peptides, and we compare the LC/MS and SFC/MS of a diverse library of druglike compounds. A total of 75.0% of the library compounds were eluted and detected by SFC/MS, while 79.4% were eluted and detected by LC/MS. Some samples provided strong peaks that appeared to be related to the purported compound contained in the sample. When these were added to the "hits", the numbers rose to 86.7 and 89.9%, respectively. A total of 3.7% of the samples were observed by SFC/MS, but not by LC/MS, and 8.1% of the samples were observed by LC/MS, but not by SFC/MS. The only compound class that appeared to be consistently detected in LC/MS, but not in SFC/MS under our conditions, consisted of compounds containing a phosphate, a phosphonate, or a bisphosphonate. The SFC/MS method was at least as durable, reliable, and user-friendly as the LC/MS method. The APCI source required less cleaning during the SFC/MS separations than it did during LC/MS.     

Screening and sequencing of glycated proteins by neutral loss scan LC/MS/MS method

Nonenzymatic protein glycation is caused by a Schiff's base reaction between the aldehyde groups of reducing sugars and the primary amines of proteins. A reversed-phase liquid chromatography method followed by a neutral loss scan mass spectrometric method was developed for the screening of glycation in proteins. The neutral loss scan was based on a unique sugar moiety neutral loss (-162 Da) that we observed in the fragmentation spectra of glycated peptides on Q-Tof type mass spectrometers. The collision energy was optimized for this neutral loss using a glycated synthetic peptide, and 20 eV was found to be the optimum collision energy. The neutral loss scan experiment was composed of two segments. In the first segment, the glycated peptides were identified based on the signature neutral loss of 162 Da when the collision energy was elevated to 20 eV. In the second segment, the glycated peptides were selected as the parent ions and fragmented at higher collision energy to break the peptide bonds. The fragmentation spectra of the selected glycated peptides revealed both the amino acid sequences and the sites of glycation. This neutral loss scan method was used to study the glycation in human serum albumin (HSA). The glycation sites in HSA were identified based on the retention time shift of glycated peptides, the mass accuracy from the MS scan, the signature neutral loss, and MS/MS information. Using this method, we were able to identify that 31 lysine residues were partially glycated from the glycated HSA sample, which has a total of 59 lysine residues.     

血液中佐匹克隆全自动固相萃取高效液相色谱质谱检测方法研究

目的:建立血液中佐匹克隆全自动固相萃取高效液相色谱-三重四级杆串联质谱(LC/MS/MS)检测方法。方法:采用全自动固相萃取进行提取,用Oasis HLB固相萃取柱,使用二氯甲烷洗脱。洗脱物浓缩后用Eclipse Plus C18柱(3.5μm×2.1×100mm)分离,LC/MS/MS分析。结果:采用本文方法检测血液中佐匹克隆的检出限为0.1ng/ml,回收率大于90.0%。结论:本方法具有操作简单、灵敏度高和重现性好等优点,可应用于血液中佐匹克隆的检验鉴定。