Enhanced characterization of complex proteomic samples using LC-MALDI MS/MS: exclusion of redundant peptides from MS/MS analysis in replicate runs

Due to the complexity of proteome samples, only a portion of peptides and thus proteins can be identified in a single LC-MS/MS analysis in current shotgun proteomics methodologies. It has been shown that replicate runs can be used to improve the comprehensiveness of the proteome analysis; however, high-intensity peptides tend to be analyzed repeatedly in different runs, thus reducing the chance of identifying low-intensity peptides. In contrast to commonly used online ESI-MS, offline MALDI decouples the separation from MS acquisition, thus allowing in-depth selection for specific precursor ions. Accordingly, we extended a strategy for offline LC-MALDI MS/MS analysis using a precursor ion exclusion list consisting of all identified peptides in preceding runs. The exclusion list eliminated redundant MS/MS acquisitions in subsequent runs, thus reducing MALDI sample depletion and allowing identification of a larger number of peptide identifications in the cumulative dataset. In the analysis of the digest of an Escherichia coli lysate, the exclusion list strategy resulted in a 25% increase in the number of unique peptide identifications in the second run, in contrast to simply pooling MS/MS data from two replicate runs. To reduce the increased LC analysis time for repeat runs, a four-column multiplexed LC system was developed to carry out separation simultaneously. The multiplexed LC-MALDI MS provides a high-throughput platform to utilize the exclusion list strategy in proteome analysis.     

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.     

Pinpointing biomarkers in proteomic LC/MS data by moving-window discriminant analysis

The identification of differential patterns in data originating from combined measurement techniques such as LC/MS is pivotal to proteomics. Although "shotgun proteomics" has been employed successfully to this end, this method also has severe drawbacks, because of its dependence on largely untargeted MS/MS sequencing and databases for statistical analyses. Alternatively, several MS-signal-based (MS/MS-independent) methods have been published that are mainly based on (univariate) Student's t-tests. Here, we present a more robust multivariate alternative employing linear discriminant analysis. Like the t-test-based methods, it is applied directly to LC/MS data, instead of using MS/MS measurements. We demonstrate the method on a number of simulated data sets, as well as on a spike-in LC/MS data set, and show its superior performance over t-tests.     

High-throughput bioanalytical LC/MS/MS determination of benzodiazepines in human urine: 1000 samples per 12 hours.

The analytical capabilities of liquid chromatography tandem mass spectrometry for sensitive and highly selective determination of target compounds in complex biological samples makes it well suited for high-throughput analysis. We report the fast separation of six benzodiazepines isolated from human urine via selected reaction monitoring liquid chromatography/mass spectrometry using short dwell times to accommodate fast-eluting chromatographic peaks. The analytes were extracted from human urine samples along with their deuterium-labeled internal standards by a simple liquid-liquid extraction in 96-well plates. Using four autosamplers coupled to one chromatographic column and one tandem mass spectrometer operated in the turbo ion spray mode with positive ion detection, 1152 samples (12 96-well plates) were analyzed in less than 12 h. Through an electronic switching box designed and constructed in-house, the autosamplers were synchronized with the mass spectrometer so that injections were made as soon as the mass spectrometer was ready to collect data. Each run required 30 s to complete with another 7-8 s for the data system to load the next data file to be collected. Chromatographic integrity and ion current response remained relatively constant for the duration of the analyses. The results show acceptable precision and accuracy and demonstrate the feasibility of using fast separations with tandem mass spectrometry for high-throughout analysis of biological samples containing multiple analytes.     

Simultaneous LC/MS/MS determination of thiols and disulfides in urine samples based on differential labeling with ferrocene-based maleimides

A method for the simultaneous determination of a series of thiols and disulfides in urine samples has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. The sample is first exposed to N-(2-ferroceneethyl)maleimide, thus leading to the derivatization of free thiol groups in the sample. After quantitative reaction and subsequent reduction of the disulfide-bound thiols by tris(2-carboxyethyl)phosphine, the newly formed thiol functionalities are reacted with ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide. The reaction products are determined by LC/MS/MS in the multiple reaction mode, and precursor ion scan as well as neutral loss scan is applied to detect unknown further thiols. The method was successfully applied to the analysis of free and disulfide-bound thiols in urine samples. Limits of detection are 30 to 110 nM, and the linear range comprises two decades of concentration, thus covering the relevant concentration range of thiols in urine samples. The thiol and disulfide concentrations were referred to the creatinine content to compensate for different sample volumes. As some calibration standards for the disulfides are not commercially available, they were synthesized in an electrochemical flow-through cell. This allowed the synthesis of hetero- and homodimeric disulfides.     

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   

Accelerated solvent extraction followed by on-line solid-phase extraction coupled to ion trap LC/MS/MS for analysis of benzalkonium chlorides in sediment samples

Benzalkonium chlorides (BACs) were successfully extracted from sediment samples using a new methodology based on accelerated solvent extraction (ASE) followed by an on-line cleanup step. The BACs were detected by liquid chromatography/ion trap mass spectrometry (LC/MS) or tandem mass spectrometry (MS/MS) using an electrospray interface operated in the positive ion mode. This methodology combines the high efficiency of extraction provided by a pressurized fluid and the high sensitivity offered by the ion trap MS/MS. The effects of solvent type and ASE operational variables, such as temperature and pressure, were evaluated. After optimization, a mixture of acetonitrile/water (6:4 or 7:3) was found to be most efficient for extracting BACs from the sediment samples. Extraction recoveries ranged from 95 to 105% for C12 and C14 homologues, respectively. Total method recoveries from fortified sediment samples, using a cleanup step followed byASE, were 85% for C12BAC and 79% for C14BAC. The methodology developed in this work provides detection limits in the subnanogram per gram range. Concentrations of BAC homologues ranged from 22 to 206 microg/kg in sediment samples from different river sites downstream from wastewater treatment plants. The high affinity of BACs for soil suggests that BACs preferentially concentrate in sediment rather than in water.     

A high-capacity LC/MS system for the bioanalysis of samples generated from plate-based metabolic screening

HPLC/MS is a linear technique characterized by serial injection and analysis of individual samples. Parallel-format high-throughput screens for druglike properties present a significant analytical challenge. Analysis speed and system ruggedness are key requirements for bioanalysis of thousands of samples per day. The tasks involved in LC/MS analysis are readily divided into three areas, sample preparation/liquid handling, LC/MS method building/sample analysis, and data processing. Several automation and multitasking strategies were developed and implemented to minimize plating and liquid handling errors, reduce dead times within the analysis cycle, and allow for comprehensive review of data. Delivering multiple samples to multiple injectors allows the autosampler time to complete its wash cycles and aspirate the next set of samples while the previous set is being analyzed. A dual-column chromatography system provides column cycling and peak stacking and allows rapid throughput using conventional LC equipment. Collecting all data for a compound into a single file greatly reduces the number of data files collected, increases the speed of data collection, allows rugged and complete review of all data, and provides facile data management. The described systems have analyzed over 40 000 samples per month for two years and have the capacity for over 2000 samples per instrument per day.     

MS/MS methodology to improve subcellular mapping of cholesterol using TOF-SIMS

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) can be utilized to map the distribution of various molecules on a surface with submicrometer resolution. Much of its biological application has been in the study of membrane lipids, such as phospholipids and cholesterol. Cholesterol is a particularly interesting molecule due to its involvement in numerous biological processes. For many studies, the effectiveness of chemical mapping is limited by low signal intensity from various biomolecules. Because of the high energy nature of the SIMS ionization process, many molecules are identified by detection of characteristic fragments. Commonly, fragments of a molecule are identified using standard samples, and those fragments are used to map the location of the molecule. In this work, MS/MS data obtained from a prototype C60(+)/quadrupole time-of-flight mass spectrometer was used in conjunction with indium LMIG imaging to map previously unrecognized cholesterol fragments in single cells. A model system of J774 macrophages doped with cholesterol was used to show that these fragments are derived from cholesterol in cell imaging experiments. Examination of relative quantification experiments reveals that m/z 147 is the most specific diagnostic fragment and offers a 3-fold signal enhancement. These findings greatly increase the prospects for cholesterol mapping experiments in biological samples, particularly with single cell experiments. In addition, these findings demonstrate the wealth of information that is hidden in the traditional TOF-SIMS spectrum.     

Time-Efficient LC/MS/MS Determination of Low Concentrations of Methylphosphonic Acid

A time-efficient protocol for quantification of methylphosphonic acid (MPA), a final hydrolysis product of nerve agents in aqueous environments, via liquid chromatography mass spectrometry is described. Chromatographic separation and mass spectrometry detection conditions are optimized to enable high sensitivity, selectivity, and accuracy of the approach and to eliminate impeding matrix effects associated with the analysis of water samples of natural origin. The developed technique is tested by analyzing the samples of natural waters that are spiked with a known quantity of MPA. With direct LC/MS/MS, the detection limit for MPA in natural waters is 10 ng/mL.     

LC/MS/MS method for quantitative determination of long-chain fatty acyl-CoAs

Long-chain acyl-CoA esters (LCACoAs) are activated lipid species that represent key substrates in lipid metabolism. The relationship between lipid metabolism disorders and type 2 diabetes has attracted much attention to this class of metabolites. This paper presents a highly sensitive and robust on-line LC/MS(2) procedure for quantitative determination of LCACoAs from rat liver. A fast SPE method has been developed without the need for time-consuming evaporation steps for sample preparation. LCACoAs were separated with high resolution using a C18 reversed-phase column at high pH (10.5) with an ammonium hydroxide and acetonitrile gradient. Five LCACoAs (C16:0, C16:1, C18:0 C18:1, C18:2) were quantified by selective multireaction monitoring using a triple quadrupole mass spectrometer in positive electrospray ionization mode. It is possible to perform a neutral loss scan of 507 for lipid profiling of complex LCACoA mixtures in tissue extracts. The method presented was validated according to ICH guidelines for quantitative determination of five LCACoAs for physiological concentrations in 100-200 mg of tissue with accuracies ranging from 94.8 to 110.8%, interrun precisions between 2.6 and 12.2%, and intrarun precisions between 1.2 and 4.4%. Due to the high sensitivity of the developed method, the amount of tissue biopsied for reliable quantification can be reduced. This may be advantageous in the quantification of LCACoAs in humans.     

In vivo deamidation characterization of monoclonal antibody by LC/MS/MS

The spontaneous nonenzymatic deamidation of glutaminyl and asparaginyl residues of peptides and proteins has been observed both in vitro and in vivo. Deamidation may change the structure and function of a peptide or protein, potentially resulting in decreased bioactivity, as well as alterations in pharmacokinetics and antigenicity of the protein pharmaceutical. Therefore, it is necessary to monitor the effect of storage and formulation conditions on deamidation of a protein drug candidate. Of particular interest is the investigation of in vivo deamidation mechanisms of protein drug candidates. Several methods are available to characterize the deamidation of peptides and proteins. We present here a LC/MS/MS method used to evaluate the deamidation of an antibody after in vivo administration. A humanized monoclonal IgG1 antibody (MAb) has several "hot spots" for spontaneous deamidation. One site, amino acid residue Asn55 located in the CDR2 region of the heavy chain, is of particular interest since deamidation at this site greatly decreases the binding activity. MAb was administered to cynomolgus monkeys by intravenous and subcutaneous routes. At various times after dosing, monkey serum was prepared and MAb captured by the immobilized antigen or a goat anti-human IgG Fcgamma antibody. The captured MAb was treated with trypsin followed by endoproteinase Glu-C. The digests were separated on RP-HPLC and analyzed by MS/MS on Q-Tof Global mass spectrometer. Using this method, we were able to determine the deamidation half-life of amino acid residue Asn55 in vivo and the ratio of the deamidated derivatives, i.e., isoAsp55 and Asp55. The method is rapid and sensitive with low-nanogram quantities of protein detected in the biological matrix.     

LC/MS/MS analyses of an oleander extract for cancer treatment

An HPLC/MS/MS method has been developed for the characterization and quantification of the cardiac glycosides oleandrin, odoroside, neritaloside and the aglycone oleandrigenin, all contained in a patented-hot-water extract of Nerium oleander L (Anvirzel). Qualitative analysis of such extracts was achieved using a hybrid tandem quadrupole time-of-flight (QqTOF) mass spectrometer. Collision-induced dissociation (CID) mass spectra of oleandrin, oleandrigenin, odoroside, and neritaloside were obtained with greater than 5 ppm mass accuracy and resolution routinely in excess of 8000 (fwhm). The detection limit for oleandrin of 20 pg (injected) was realized when the precursor-to-product ion transition, m/z 577 --> 373, was monitored. We have also applied the analytical method to the determination of oleandrin, oleandrigenin, neritaloside, and odoroside in human plasma following an intramuscular injection of Anvirzel.     

Integration of two-dimensional LC-MS with multivariate statistics for comparative analysis of proteomic samples

LC-MS-based proteomics requires methods with high peak capacity and a high degree of automation, integrated with data-handling tools able to cope with the massive data produced and able to quantitatively compare them. This paper describes an off-line two-dimensional (2D) LC-MS method and its integration with software tools for data preprocessing and multivariate statistical analysis. The 2D LC-MS method was optimized in order to minimize peptide loss prior to sample injection and during the collection step after the first LC dimension, thus minimizing errors from off-column sample handling. The second dimension was run in fully automated mode, injecting onto a nanoscale LC-MS system a series of more than 100 samples, representing fractions collected in the first dimension (8 fractions/sample). As a model study, the method was applied to finding biomarkers for the antiinflammatory properties of zilpaterol, which are coupled to the beta2-adrenergic receptor. Secreted proteomes from U937 macrophages exposed to lipopolysaccharide in the presence or absence of propanolol or zilpaterol were analysed. Multivariate statistical analysis of 2D LC-MS data, based on principal component analysis, and subsequent targeted LC-MS/MS identification of peptides of interest demonstrated the applicability of the approach.     

Collection, storage, and filtration of in vivo study samples using 96-well filter plates to facilitate automated sample preparation and LC/MS/MS analysis

The benefits of high-throughput bioanalysis within the pharmaceutical industry are well established. One of the most significant bottlenecks in bioanalysis is transferring in vivo-generated study samples from their collection tubes during sample preparation and extraction. In most cases, the plasma samples must be stored frozen prior to analysis, and the freeze/thaw (F/T) process introduces thrombin clots that are capable of plugging pipets and automated liquid-transfer systems. A new approach to dealing with this problem involves the use of Ansys Captiva 96-well 20-microm polypropylene filter plates to collect, store frozen, and filter plasma samples prior to bioanalysis. The samples are collected from the test subjects, and the corresponding plasma samples are placed directly into the wells of the filter plate. Two Duoseal (patent pending) covers are used to seal the top and bottom of the plate, and the plate is stored at down to -70 degrees C. Prior to sample analysis, the seals are removed and the plate is placed in a 96-well SPE manifold. As the plasma thaws, it passes (by gravity or mild vacuum) through the polypropylene filter into a 96-well collection plate. A multichannel pipet or automated liquid-transfer system is used to transfer sample aliquots without fear of plugging. A significant advantage of this approach is that, unlike other methods, issues related to incomplete pipetting are virtually eliminated. The entire process is rapid since thawing and filtering take place simultaneously, and if a second F/T cycle is required for reanalysis, it is not necessary to refilter the samples (additional clotting was not observed after three F/T cycles). This technique was tested using monkey, rat, and dog plasma and sodium heparin and EDTA anticoagulants. To assess the possibility of nonspecific binding to the polypropylene filter, a variety of drug candidates from diverse drug classes were studied. Validation data generated for two Lilly compounds from distinct classes, before and after filtering, are presented in this paper as practical examples of this technique. While LC/MS/MS is the primary method of bioanalysis in our laboratory, the technique presented in this paper is applicable to other forms of detection as well.     

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.     

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.     

Separation of cis-trans phospholipid isomers using reversed phase LC with high resolution MS detection

The increased presence of synthetic trans fatty acids into western diets has been shown to have deleterious effects on physiology and raising an individual's risk of developing metabolic disease, cardiovascular disease, and stroke. The importance of these fatty acids for health and the diversity of their (patho) physiological effects suggest that not only should the free trans fatty acids be studied but also monitoring the presence of these fats into the side chains of biological lipids, such as glycerophospholipids, is also essential. We developed a high resolution LC-MS method that quantitatively monitors the major lipid classes found in biospecimens in an efficient, sensitive, and robust manner while also characterizing individual lipid side chains through the use of high energy collisional dissociation (HCD) fragmentation and chromatographic alignment. We herein show how this previously described reversed phase method can baseline separate the cis-trans isomers of phosphatidylglycerol and phosphatidylcholine (PC) with two 18:1 side chains, in both positive and negative mode, as neat solutions and when spiked into a biological matrix. Endogenous PC (18:1/18:1)-cis and PC (18:1/18:1)-trans isomers were examined in mitochondrial and serum profiling studies, where rats were fed diets enriched in either trans 18:1 fatty acids or cis 18:1 fatty acids. In this study, we determined the cis:trans isomer ratios of PC (18:1/18:1) and related this ratio to dietary composition. This generalized LC-MS method enables the monitoring of trans fats in biological lipids in the context of a nontargeted method, allowing for relative quantitation and enhanced identification of unknown lipids in complex matrixes.     

Use of peptide retention time prediction for protein identification by off-line reversed-phase HPLC-MALDI MS/MS

A new algorithm, sequence-specific retention calculator, was developed to predict retention time of tryptic peptides during RP HPLC fractionation on C18, 300-A pore size columns. Correlations of up to approximately 0.98 R2 value were obtained for a test library of approximately 2000 peptides and approximately 0.95-0.97 for a variety of real samples. The algorithm was applied in conjunction with an exclusion protocol based on mass (15 ppm tolerance) and retention time (2-min tolerance for 0.66% acetonitrile/min gradient), MART criteria to significantly reduce the instrument time required for complete MS/MS analysis of a digest separated by RP HPLC. This was confirmed by reanalyzing the set of HPLC-MALDI MS/MS data with no loss in protein identifications, despite the number of virtually executed MS/MS analyses being decreased by 57%.     

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.