Polyphenol identification based on systematic and robust high-resolution accurate mass spectrometry fragmentation

High-mass resolution multi-stage mass spectrometry (MS(n)) fragmentation was tested for differentiation and identification of metabolites, using a series of 121 polyphenolic molecules. The MS(n) fragmentation approach is based on the systematic breakdown of compounds, forming a so-called spectral tree. A chip-based nanoelectrospray ionization source was used combined with an ion-trap, providing reproducible fragmentation, and accurate mass read-out in an Orbitrap Fourier transform (FT) MS enabling rapid assignment of elemental formulas to the molecular ions and all fragment ions derived thereof. The used protocol resulted in reproducible MS(n) fragmentation trees up to MS(5). Obtained results were stable over a 5 month time period, a concentration change of 100-fold, and small changes in normalized collision energy, which is key to metabolite annotation and helpful in structure and substructure elucidation. Differences in the hydroxylation and methoxylation patterns of polyphenolic core structures were found to be reflected by the differential fragmentation of the entire molecule, while variation in a glycosylation site displayed reproducible differences in the relative intensities of fragments originating from the same aglycone fragment ion. Accurate MS(n)-based spectral tree data are therefore a powerful tool to distinguish metabolites with similar elemental formula, thereby assisting compound identification in complex biological samples such as crude plant extracts.     

Very high pressure gradient LC/MS/MS

A very high pressure liquid chromatography (VHPLC) system was constructed by modifying a commercially available pump in order to achieve pressures in excess of 1,200 bar (17,500 psi). A computer-controlled low-pressure mixer was used to generate solvent gradients. Protein digests were rapidly analyzed by reversed-phase VHPLC with linear solvent gradients coupled to either a tandem mass spectrometer using electrospray ionization or a UV/visible detector. The separations were performed at pressures ranging from 790 (11,500 psi) to 930 bar (13,500 psi) in 22-cm-long capillary columns packed with C18-modified 1.5-microm nonporous silica particles. A digest of bovine serum albumin (BSA) was analyzed by the VHPLC system connected to a mass spectrometer in MS mode. An analysis of 12.5 fmol of sample gave signal-to-noise ratios of tryptic peaks greater than 10:1 in the base peak plot mass chromatogram. This system was also used to analyze a proteolytic digest of a rat liver protein excised from a 2-D gel separation of a liver tissue lysate. For this analysis, the mass spectrometer was set up to perform data-dependent scanning (automated switching from MS mode to MS/MS mode when a peak was detected) for peptide sequencing and protein identification by database searching. The results of this analysis are compared to an analysis performed on the same sample using the nanoelectrospray-MS/MS technique. Though both techniques were able to identify the unknown protein, the VHPLC method gave twice as many sequenced peptides as nanoelectrospray and improved the signal-to-noise ratio of the spectra by at least a factor of 10. Direct comparisons with nanoelectrospray for MS and MS/MS data acquisition from a BSA digest were made. These comparisons show enhancements of greater than 20-fold for VHPLC over nanoelectrospray. In addition, the VHPLC/MS/MS data acquisition was accomplished in an automated manner.     

Quantifying phthalate metabolites in human meconium and semen using automated off-line solid-phase extraction coupled with on-line SPE and isotope-dilution high-performance liquid chromatography--tandem mass spectrometry

We developed an analytical method using off-line solid-phase extraction (SPE) coupled with on-line SPE and isotope-dilution high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine the concentrations of phthalate metabolites in human meconium and in semen. First, we used off-line SPE to remove interfering proteins and other biomolecules from the samples. Then, we preconcentrated the phthalate metabolites in the extract using on-line SPE before measuring them by HPLC-MS/MS. For most of the analytes, the limits of detection ranged between 0.2 and 0.7 ng/g for meconium and between 0.3 and 0.7 ng/mL for semen. The recovery after off-line SPE varied for most analytes between 65 and 99% at concentrations ranging from 3.0 to 30.0 ng/mL in semen and between 67 and 103% at concentrations ranging from 2.0 to 10.0 ng/mL in meconium. Precision measured by the relative standard deviation ranged from 3.2 to 19.1% for intraday and from 3.9 to 18.6% for interday. We validated this novel approach--which is applicable to other biological matrixes, including serum and breast milk--on spiked samples and on five meconium samples and one pooled semen sample from people with no known occupational exposure to phthalates.     

Controlled protein precipitation in combination with chip-based nanospray infusion mass spectrometry. An approach for metabolomics profiling of plasma

Liquid chromatography-mass spectrometry (LC-MS) is a common method for profiling biological samples in metabolomics. However, LC-MS data of metabolomic studies are often affected by high noise levels, retention time shifts, and high variability in signal intensities. With a new chip-based nanoelectrospray source it becomes possible to directly infuse complex biological samples such as plasma without any chromatographic separation beforehand. In combination with highly diluted samples and long data acquisition times, the parallel analysis of hundreds of compounds is now possible. In a proof-of-concept study, 10 human plasma samples from females and males were analyzed with the intention to separate the two groups by their different metabolomes. The reproducibility was so high that statistical analysis of the data could be performed without prior normalization. Two groups of female and male samples were separated by a supervised machine learning algorithm, principal component analysis, and hierarchical clustering. Peaks contributing to the group separation were characterized by accurate mass measurement and MS-MS fragmentation and by spiking experiments. The feasibility of direct sample infusion using the new chip-based nanoelectrospray device opens a new dimension for the rapid parallel analysis of complex biological mixtures.     

Analysis of organoselenium and organic acid metabolites by laser desorption single photon ionization mass spectrometry

A method for analyzing organoselenium and organic acid metabolites using laser desorption from graphite surfaces coupled to vacuum ultraviolet single photon ionization mass spectrometry (LD/SPI MS) is described. The 1-10-fmol sensitivity and linear dynamic range allows quantitative detection of selenomethionine, trimethylselenonium ion, methylselenogalactosamine, and 1beta-methylseleno-N-acetyl-D-galactosamine in complex biological samples such as human urine. In addition, common urinary metabolites such as tartronic, glutaric, orotic, uric, suberic, and hydroxyhippuric acids, are readily detected. Screening and quantitative detection of these organoselenium and organic acid metabolites is achieved within minutes. The results are also consistent with those obtained using high-performance liquid chromatography tandem mass spectrometry techniques. The study demonstrates the viability of matrix-free LD/SPI MS for molecular characterization and quantitative analysis of biological metabolites in the m/z 10-500 range that are present in complex biological fluids.     

Capillary column switching restricted-access media-liquid chromatography-electrospray ionization-tandem mass spectrometry system for simultaneous and direct analysis of drugs in biofluids

Capillary online restricted-access media-liquid chromatography-electrospray ionization-tandem mass spectrometry (RAM-LC-ESI-MS/MS) for direct analysis of drugs and metabolites spiked in biological fluids was developed. Using a column switching setup it was possible to perform effective sample preparation and analysis of raw biological fluids (plasma and urine) without matrix effects in the electrospray mass spectrometric detection step. The peak focusing efficiency of the extraction column was more effective in backflush compared to foreflush mode. The system was able to concentrate diminished samples of polar drugs and their metabolites reaching quantifiable results as low as 1 ng/mL utilizing a sample volume of only 333 nL of biofluids. New column hardware was developed to circumvent clogging problems experienced with plasma injections. The glass fiber filter frit, which is commonly used, was replaced with a short piece of 20 microm i.d. fused silica capillary. The extraction columns were able to handle up to 60 injections and showed a high loading capacity, making the saturation of the MS detector the limiting factor on the linear dynamic range. The simultaneous separation and detection of 10 drugs and metabolites was obtained in 8 min of analysis, including the online sample preparation and enrichment step.     

Reduction of signal suppression effects in ESI-MS using a nanosplitting device.

Electrospray ionization mass spectrometry is a valuable tool in the identification and quantification of drug metabolites in biological fluids. However, there are many instances where matrix components present in these fluids interfere with analyte detection and prevent the acquisition of accurate or complete results. In some instances, the matrix can suppress ionization to such an extent that analytes are completely undetectable by MS. In this work, we investigate how ionization and ion-transfer efficiencies are affected by drastically reducing the flow into the MS. A postcolumn concentric flow-splitting device was constructed to allow the measurement of analyte signal and ionization suppression across a range of flow rates (0.1-200 microL/min). Using this device, the effects of flow rate on signal intensity and ionization suppression were measured in analytical experiments that included flow injection analysis MS, postcolumn addition LC-MS, and on-line LC-MS analysis of metabolites generated from rat liver microsomes. The device used to deliver 0.1 microL/min flows is referred to as a nanosplitter because it achieved high split ratios (2000:1), producing flow rates comparable to those observed in nanoelectrospray. The nanosplitter maintained chromatographic integrity with high fidelity and allowed the direct comparison of analyte signal across a range of flow rates (0.1-200 microL/min). A significant improvement in concentration and mass sensitivity as well as a reduction in signal suppression is observed when the performance at 200 versus 0.1 microL/min flow rate is compared. Using this specially designed concentric splitting device, the advantages of ultralow flow ESI were easily exploited for applications employing large bore chromatography.     

Nanoscale ion-pair reversed-phase HPLC-MS for sensitive metabolome analysis

In this article, we introduce a method using nanoscale ion-pair reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC) hyphenated to nanoelectrospray ionization high-resolution mass spectrometry (nano-ESI-HRMS) to separate and identify metabolites in cell extracts. Separation of metabolites was performed on a 100 μm i.d. C18 column with tributylamine (TBA) as the ion-pairing reagent and methanol as the eluent. Basic pH (9.4) of the mobile phase was critical to achieve sufficient retention and sharp metabolite elution at a low concentration of TBA (1.7 mM). Limits of detection were determined for 54 standards with an LTQ-Orbitrap mass spectrometer to be in the upper attomole to low femtomole range for key metabolites such as nucleotides, phosphorylated sugars, organic acids, and coenzyme A thioesters in solvent as well as in a complex matrix. To further evaluate the method, metabolome analysis was performed injecting different amounts of biomass of the methylotroph model organism Methylobacterium extorquens AM1. A (12)C/(13)C labeling strategy was implemented to improve metabolite identification. Analysis of three biological replicates performed with 1.5 ng of cell dry weight biomass equivalents resulted in the identification of 20 ± 4 metabolites, and analysis of 150 ng allowed identifying 157 ± 5 metabolites from a large spectrum of metabolite classes.     

Identification of selenium-containing glutathione S-conjugates in a yeast extract by two-dimensional liquid chromatography with inductively coupled plasma MS and nanoelectrospray MS/MS detection

An approach for the identification of unknown selenium-containing biomolecules was developed, enabling the identification of selenodiglutathione (GS-Se-SG) and the mixed selenotrisulfide of glutathione and cysteinylglycine (GS-Se-SCG) in aqueous yeast extracts. The method consists of two-dimensional liquid chromatography, inductively coupled plasma mass spectrometry (ICPMS) and nanoelectrospray tandem mass spectrometry. Analytes were separated by size-exclusion chromatography followed by preconcentration and separation on a porous graphitic carbon HPLC column. The HPLC effluent was monitored for selenium by ICPMS, and two selenium-containing fractions were isolated and analyzed by nanoelectrospray MS. The nanoelectrospray technique has a low sample consumption of approximately 80 nL/min, enabling a preconcentration of the sample to a few microliters. Mass spectra of the two fractions showed the characteristic Se isotopic pattern centered at m/z 693.1 and 564.0 for the [M + H]+ 80Se ions. MS/MS spectra of adjacent parent ions confirmed the presence of Se. The two selenium species were identified as GS-Se-SG and GS-Se-SCG by collision induced dissociation (CID). The accurately measured masses of the most abundant 691 and 693 u parent ions are in good agreement (differences = 3 ppm) with the theoretical masses. To our knowledge, this is the first identification of GS-Se-SG and GS-Se-SCG in biological matrixes by MS/MS.     

In situ molecular analysis of plant tissues by live single-cell mass spectrometry

We report the development of a rapid, direct molecular analysis of live, single plant cells viewed under a video microscope in their natural environment. A nanoelectrospray tip was used to extract the contents of a single leaf, stem, or petal cell from Pelargonium zonale, and the samples were analyzed on an Orbitrap mass spectrometer by nanoelectrospray ionization. Around a thousand m/z peaks belonging to metabolites and other compounds in each sample were obtained and processed by using statistical tools to find the cell specific molecular peaks. Hybrid high-resolution mass spectrometry analysis was performed to confirm the structure of specific metabolites from the analyzed samples. This method is useful for identifying specific molecules in live single cells from plant tissue and will allow different cell types and stages from different sites in the plant to be compared with morphological observations.     

A four-column parallel chromatography system for isocratic or gradient LC/MS analyses

A novel approach to parallel liquid chromatography/ tandem mass spectrometry (LC/MS/MS) analyses for pharmacokinetic assays and for similar quantitative applications is presented. Modest modifications render a conventional LC/MS system capable of analyzing samples in parallel. These modifications involve the simple incorporation of three valves and four LC columns into a conventional system composed of one binary LC pumping system, one autosampler, and one mass spectrometer. An increase in sample throughput is achieved by staggering injections onto the four columns, allowing the mass spectrometer to continuously analyze the chromatographic window of interest Using this approach, the optimized run time is slightly greater than the sum of the widths of the desired peaks. This parallel chromatography unit can operate under both gradient and isocratic LC conditions. To demonstrate the utility of the system, atorvastatin, five of its metabolites, and their deuterated internal standards (IS) were analyzed using gradient elution chromatography conditions. The results from a prestudy assay evaluation (PSAE) tray of standards and quality control (QC) samples from extracted spiked human plasma are presented. The relative standard deviation and the accuracy of the QC samples did not exceed 8.1% and 9.6%, respectively, which is well within the acceptance criteria of the pharmaceutical industry. For this particular analysis, the parallel chromatography system decreased the overall run time from 4.5 to 1.65 min and, therefore, increased the overall throughput by a factor of 2.7 in comparison to a conventional LC/MS/MS analytical method.     

Chip-based capillary electrophoresis/mass spectrometry determination of carnitines in human urine

A chip-based capillary electrophoresis/mass spectrometry (CE/MS) system is described for the CE separation and on-line electrospray detection of carnitine and selected acylcarnitines from mixtures of analytical standards as well as extracts of fortified human urine. Chip-based CE/MS experiments in two different laboratories were carried out using a triple-quadrupole mass spectrometer and a quadrupole time-of-flight (QTOF) mass spectrometer, respectively. The glass chips used with both systems were comparably equipped with a microfabricated capillary electrophoresis (CE) channel but with different electrosprayers. The quadrupole chip-based CE/MS experiments employed a miniature coupled microsprayer, which allowed coupling of the microelectrospray process via a micro liquid junction at the exit of the CE capillary channel. Selected ion monitoring (SIM) CE/MS experiments were employed for all of the quadrupole CE/MS work. The QTOF CE/MS full-scan single MS and MS/MS experiments were carried out in another laboratory using accurate mass measurement TOF mass spectrometry techniques. The electrospray process that was employed with the QTOF system differed in that an inserted nanoelectrospray capillary needle was carefully affixed into a flat-bottomed hole that was aligned with the CE channel exit orifice. SIM CE/MS using the described quadrupole system provided acceptable ion current electropherograms from fmole levels from analytical standard solutions of carnitine and acylcarnitines that were manually injected (loaded) onto the chip. In addition, the corresponding electropherograms for human urine fortified with the target carnitine and acylcarnitines at a 10-20 microg/mL (35-124 microM) level were obtained via SIM CE/MS techniques. The measured CE separation efficiency for the SIM CE/MS electropherograms was determined to be 2860 plates (peak width at half-height method or N = 5.54(T/WO.5(2)), and carnitine and three acylcarnitines were separated in less than 48 s. In contrast, using quadrupole-TOF technologies, the same samples could be diluted by a factor of 2-4 to obtain a comparable detector response for the target compounds. In the full-scan, single mass analyzer mode (m/z 150-500), the CE separation efficiency was measured to be 2600 plates, but mass measurement accuracy was less than 5.0 ppm for the quaternary cations. In the CE/MS/MS mode, full-scan collision-induced dissociation (CID) mass spectra were obtained with a mass accuracy of < or =10 ppm for the higher mass ions and < or =27 ppm for the lower mass product ions. These results demonstrate the feasibility for on-chip CE separation and electrospray mass spectrometric detection for these important compounds in synthetic mixtures, as well as in human urine extracts.     

Stable-isotope trapping and high-throughput screenings of reactive metabolites using the isotope MS signature

A highly efficient method has been developed to detect and identify reactive metabolites, using stable-isotope trapping combined with ESI-MS/MS neutral loss scanning. A mixture of glutathione (GSH, gamma-glutamylcysteinylglycine) and the stable-isotope labeled compound (GSX, gamma-glutamylcysteinylglycine-(13)C(2)-(15)N) was used at an equal molar ratio to trap reactive metabolites generated in microsomal incubations. Samples resulting from incubations were cleaned and concentrated by SPE, followed by LC-MS/MS analyses using constant neutral loss scanning for 129 Da (the gamma-glutamyl moiety) to detect formed GSH conjugates. Unambiguous identification of glutathione adducts was greatly facilitated by the presence of a unique MS signature of a prominent isotopic doublet that differs in mass by 3 Da. Further structural characterization of conjugates was achieved with high confidence by subsequently acquiring MS/MS spectra that were featured by neutral losses of 75 and 129 Da for GSH adducts and 78 and 129 Da for isotopic GSX adducts. The reliability of this method was vigorously validated using a number of compounds known to form reactive metabolites. Superior sensitivity was demonstrated by the capability of the current approach to identify reactive metabolites at low abundance. Because of the unique isotopic MS signature, ultrafast analyses of reactive metabolites were accomplished by direct injection of cleaned samples into mass spectrometers for neutral loss scanning. More importantly, this study has demonstrated the feasibility of the current method for completely automated detection of reactive metabolites via computer-assisted pattern recognition.     

Capillary LC-MS for high sensitivity metabolomic analysis of single islets of Langerhans

Reversed-phase, packed capillary liquid chromatography interfaced by electrospray ionization to mass spectrometry was explored as an analytical method for determination of metabolites in microscale tissue samples using single islets of Langerhans as a model system. With the use of a 75 microm inner diameter column coupled to a quadrupole ion trap mass spectrometer in full scan mode, detection limits of 0.1-33 fmol were achieved for glycoloytic and tricarboxylic acid cycle metabolites. Reproducible processing of islets for analysis with little loss of metabolites was performed by rapid freezing followed by methanol-water extraction. The method yielded 20 microL of extract of which just 15 nL was injected suggesting the potential for performing multiple assays on the same islet. Approximately 200 presumed metabolites could be detected, of which 22 were identified by matching retention times and MS/MS spectra to standards. Relative standard deviations for peak detection was from 7 to 18% and was unaffected by storage for up to 11 days. The method was used to detect changes in metabolism associated with increasing extracellular islet glucose concentration from 3 to 20 mM yielding results largely consistent with known metabolism of islets. Because most previous studies of islet metabolism have only observed a few compounds at once and require far more tissue, this measurement method represents a significant advance for studies of metabolism of islets and other microscale samples.     

Dual conductivity/amperometric detection system for microchip capillary electrophoresis

The performance characteristics and advantages of a new dual electrochemical microchip detection system based on simultaneous conductivity and amperometric measurements are described. The system relies on the combination of a contactless conductivity detector with an end-column thick-film amperometric detector. Such coupling of the conductivity and amperometric detection modes in a single separation channel greatly enhances the sample characterization to offer simultaneous measurements of both ionic and electroactive species, improved reproducibility, and confirmation of peak identity. The simultaneous measurement of nitroaromatic and ionic explosives is used for demonstrating the ability to detect both electroactive and ionic species. Major improvements are also observed for analytes responding at both detectors. For example, the generation of dual response ratios can be used to improve the reproducibility and confirm the peak identity/integrity. Such dual response ratios reflect the distinct redox and conductivity properties of the individual analytes. The independence of the two detectors is reflected in the absence of "cross-talk" effects. The behavior of the dual detector is comparable with those of the individual detectors. Such a dual electrochemical detection system is easy to implement and requires inherently portable low-cost instrumentation.     

Integrated microfluidic device for mass spectrometry-based proteomics and its application to biomarker discovery programs

The present investigation describes the analytical performances of a microfluidic device comprising an enrichment column, a reversed-phase separation channel, and a nanoelectrospray emitter embedded altogether in polyimide layers. This configuration minimizes transfer lines and connections and reduces postcolumn peak broadening and dead volumes. This compact and versatile modular nanoLC-chip system was interfaced to both ion trap and time-of-flight mass spectrometers, and its analytical potentials were evaluated in the context of proteomics applications. The figures of merit of this system in terms of peak capacity, reproducibility, sensitivity, and linear dynamic range of peptide detection were determined using tryptic digests of complex protein extracts including albumin- and immunoglobulin-depleted rat plasma samples. The analysis of peak profiles for more than 600 peptide ions reproducibly detected across replicate nanoLC-chip-MS runs (n = 10) indicated that this system provided good reproducibility of retention time and peak intensity with RSD values of less than 0.5 and 9.1%, respectively. Variation in peptide abundance as low as 2-fold changes was identified for spiked tryptic digests present at levels of 2-5 fmol in plasma samples. Sensitivity measurements were performed on dilution series of protein digests spiked into rat plasma samples and provided a detection limit of 1-5 fmol. The modular concept of the microfluidic systems also facilitated the integration of two-dimensional chromatography (strong cation exchange/C18) thereby increasing the sample loading and selectivity of the nanoLC-chip-MS system. The application of this integrated device was evaluated for complex rat plasma samples to compare the number of protein identifications obtained using one- and two-dimensional nanoLC-chip-MS/MS.     

Annotation of the human adult urinary metabolome and metabolite identification using ultra high performance liquid chromatography coupled to a linear quadrupole ion trap-orbitrap mass spectrometer

Metabolic profiles of biofluids obtained by atmospheric pressure ionization mass spectrometry-based technologies contain hundreds to thousands of features, most of them remaining unknown or at least not characterized in analytical systems. We report here on the annotation of the human adult urinary metabolome and metabolite identification from electrospray ionization mass spectrometry (ESI-MS)-based metabolomics data sets. Features of biological interest were first of all annotated using the ESI-MS database of the laboratory. They were also grouped, thanks to software tools, and annotated using public databases. Metabolite identification was achieved using two complementary approaches: (i) formal identification by matching chromatographic retention times, mass spectra, and also product ion spectra (if required) of metabolites to be characterized in biological data sets to those of reference compounds and (ii) putative identification from biological data thanks to MS/MS experiments for metabolites not available in our chemical library. By these means, 384 metabolites corresponding to 1484 annotated features (659 in negative ion mode and 825 in positive ion mode) were characterized in human urine samples. Of these metabolites, 192 and 66 were formally and putatively identified, respectively, and 54 are reported in human urine for the first time. These lists of features could be used by other laboratories to annotate their ESI-MS metabolomics data sets.     

Parallel electrophoretic depletion, fractionation, concentration, and desalting of 96 complex biological samples for mass spectrometry

The preparation of complex biological samples for high-throughput mass spectrometric analyses remains a significant bottleneck, limiting advancement of the capabilities of mass spectrometry (MS) and ultimately limiting development of novel clinical assays. The removal of interfering species (e.g., salts, detergents, and buffers), concentration of dilute analytes, and the reduction of sample complexity are required in order to maximize the quality of resultant MS data. This study describes a novel sample preparation method that makes use of electrophoresis to prepare complex biological samples for high-throughput MS analysis. The method provides for integration of key sample preparation steps, including depletion, fractionation, desalting, and concentration. The prepared samples are captured onto a monolithic reversed-phase capture target that can be analyzed directly by a mass spectrometer. Up to 96 individual samples are simultaneously prepared for MS analysis in under 1 h. For standard proteins added to serum, this method provides femtomole level sensitivity and reproducible label-free detection (coefficient of variation <30%). This study demonstrates that this electrophoretic sample preparation system permits high-throughput sample preparation for mass spectrometric analysis of complex biological samples, such as serum, plasma, and tissue extracts.