Liquid chromatography-Fourier Transform Ion Cyclotron Research Articles

A comprehensive quality evaluation strategy of Shensong Yangxin capsules based on qualitative, fingerprint and quantitative analyses.

Shensong Yangxin capsule (SSYXC), an effective Chinese patent medicine, has been recorded in the Chinese Pharmacopeia, mainly for the treatment of coronary heart disease and ventricular premature beat. To further complete the quality evaluation of SSYXC, a comprehensive analysis strategy was established. Firstly, the components of SSYXC were qualitatively analysed using ultra-high- performance liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry. A total of 134 compounds were identified or tentatively characterized. Additionally, the fingerprint of SSYXC was established by HPLC, and the similarity of 10 batches of SSYXC was elucidated by similarity analysis. The result indicated that the consistency of chemical composition is good. Finally, to enhance the quality control of SSYXC, according to the results of the fingerprint analysis, the contents of the seven active components was determined, comprising morroniside, loganin, paeoniflorin, salvianolic acid B, palmatine hydrochloride, berberine hydrochloride and tanshinone IIA. In conclusion, the established method, comprising identification of components, fingerprint analysis and quantification of multicomponents, can be sensitively and comprehensively applied to the quality evaluation of SSYXC, which can provide chemical ingredients bases for quality control and the pharmacodynamic mechanism of SSYXC, which could serve as a benchmark for controlling the quality of other Chinese patent medicines.

Chemical diversity of dissolved organic matter revealed by ultra performance liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry

Dissolved organic matter (DOM) is a highly complex and heterogeneous mixture that exists in various environments, including rivers, oceans, soils, and atmospheric aerosols. DOM plays a crucial role in biogeochemical cycles and significantly influences the environment by regulating water quality, changing the climate, and transporting pollutants. Therefore, clarifying the detailed molecular composition of DOM is essential to obtain a better understanding of its physical and chemical properties, thereby enabling further elucidation of its biogeochemical behavior. In this study, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) combined with quadrupole detection (QPD) was used to conduct the online ultra performance liquid chromatography (UPLC)-MS analysis of DOM in water, aerosol, and soil samples collected in Tianjin, China. The samples were extracted with pure water and filtered through a glass fiber membrane (0.45 μm). The DOM in the samples was then enriched by solid-phase extraction (SPE) and redissolved in water-acetonitrile (1∶1, v/v) at mass concentration of 200 mg/L for the LC-MS experiments. The mobile phases used for UPLC were water containing 0.1% (v/v) formic acid (A) and acetonitrile containing 0.1% (v/v) formic acid (B). The gradient elution procedure was as follows: 0-5 min, 0B; 5-11 min, 0B-95%B; 11-25 min, 95%B; 25-28 min, 95%B-0B; 28-30 min, 0B. The flow rate was 0.1 mL/min, and the injection volume was 10 μL. The UV wavelength was set at 274 nm. MS detection was performed in negative electrospray ionization (ESI(-)) mode with a capillary voltage of 5.0 kV, and the MS data were collected in broadband (m/z 150-1000) and QPD modes. The transient data size was set to 2M, the free induction decay signal length was 0.74 s, and the ion accumulation time was 0.030 s. Four chromatographic peaks were observed in the chromatograms. The first peak was identified as salt adduct compounds containing sodium formate. The three other peaks contained complex components, such as oxygen-rich, unsaturated tannin-like compounds, as well as low-oxygen, highly saturated lignin-like and protein/amino-like compounds. UPLC-FT-ICR MS was suitable for assigning the detailed elemental compositions of the DOM samples. UPLC effectively improved the ionization efficiency of difficult-to-ionize compounds and enhanced the detection accuracy of MS. Indeed, MS peaks with a mass difference of as small as 3.4 mDa were well identified. A total of 12027, 15593, and 8029 peaks in the mass spectra of the water, aerosol, and soil samples, respectively, were assigned to known elemental formulae. Peaks Ⅱ and Ⅲ were hydrophilic components mainly including CHNO and CHO compounds. Compared with peak Ⅱ, peak Ⅲ exhibited a significant increase in CHNOS and CHOS, indicating that UPLC exerted a certain separation effect on these compounds. Furthermore, the aerosol samples contained a higher concentration of sulfur-containing compounds than the water and soil samples, primarily because of the abundance of organic sulfates present in atmospheric and cloud water. Data processing and graphic visualization revealed that the unique components in the water samples mainly appeared in the area of 0.1<O/C<0.5 and 1.0 <H/C<1.7. The compounds detected were low-oxygen and highly condensed lignin-like compounds. The unique components in the aerosol samples appeared in the area of 0.4<O/C<1.0 and 1.5<H/C<2.0, and were classified as carbohydrates. The unique components in the hydrophilic fraction of the soil samples were found in the area of 0.6<O/C<1.0 and 0.5<H/C<1.0, and were determined to be tannin-like compounds. By contrast, the components in the hydrophobic fraction were similar to those found in the water samples and appeared in the region containing lignin-like compounds. In summary, this study proposed a novel analytical protocol to characterize DOM from different ecosystems using UPLC-FT-ICR MS. This method could separate DOM components using UPLC with eluents of different polarities and analyze them using high-resolution FT-ICR MS to reveal their molecular compositions and possible chemical types. This protocol offers solid technical support for the comprehensive profiling of DOM at the molecular level.

Rapid characterization of the chemical constituents of Duzhong Jiangya tablet by HPLC coupled with Fourier transform ion cyclotron resonance mass spectrometry.

Duzhong Jiangya tablet is a hypotensive drug. In this study, high-performance liquid chromatography-Fourier transform-ion cyclotron resonance-mass spectrometry technology was used to quickly identify its chemical composition. SinoChrom ODS-BP column (250mm × 4.6mm, 5 μm) was used. The mobile phase was acetonitrile(A)-0.1% formic acid solution(B). The flow rate was 1mL/min. Extracted ion chromatogram was used to analyze the samples in positive and negative ion modes. Based on the accurate mass spectrometry information (such as quasi-molecular ions and fragment ions) obtained from the instrument, combined with reference compounds and literature, the chemical composition of Duzhong Jiangya Tablets was identified. A total of 131 compounds were identified, including four types of penylpropanoids, six types of phenylethanoid glycosides, 10 types of organic acids, 14 types of iridoids, 12 types of lignans, 18 types of alkaloids, seven types of coumarins, and 60 kinds of flavonoids. This established method can quickly and efficiently identify chemical constituents in Duzhong Jiangya tablet, lay a foundation for the research on the efficacy and quality of this traditional Chinese medicine, and provide a reference for the characterization of the chemical constituents of other traditional Chinese medicine preparations.

Metabolome profile of Negi-Nira chive, an interspecies hybrid of green spring onion (Allium fistulosum) and Chinese chive (A. tuberosum).

Metabolome analysis of flavored vegetables, green spring onion (Allium fistulosum), Chinese chive (A. tuberosum), and their interspecies hybrid Negi-Nira chive, was conducted using liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry, with ca. 2 ppm mass accuracy. Ion peaks in the chromatograms of four biological replicates of the vegetable leaves were processed using the alignment software PowerGet for metabolite comparison, from which we obtained the potential chemical formulae. In total, 860 ion peaks were reproducibly detected; of these, 506, 525, and 336 peaks were found in the hybrid, A. tuberosum, and A. fistulosum, respectively. There were 130 peaks specific to the hybrid; from these, 31 metabolites were annotated by searching compound databases. The sulfur-containing compounds and flavonoids were further analyzed using bioinformatics, to examine the sulfur metabolism of Allium volatiles and the flavonoid pathways in these species. In conclusion, our metabolome analysis of this interspecies hybrid and its parents provides a unique opportunity to elucidate their metabolic background.

Human Dendritic Cells with Th2-Polarizing Capacity: Analysis Using Label-Free Quantitative Proteomics

Background: Dendritic cells (DCs) are the sentinels of the immune system. Upon recognition of a pathogen, they mature and migrate to draining lymph nodes to prime and polarize T cell responses. Although it is known that helminths and helminth-derived molecules condition DCs to polarize T helper (Th) cells towards Th2, the underlying mechanisms remain incompletely understood. Objectives: The aim of this study was to conduct a proteome analysis of helminth antigen-stimulated DCs in order to gain more insight into the cellular processes associated with their ability to polarize immune responses. Methods: We analyzed the maturation and polarization of monocyte-derived DCs from 9 donors at 2 different time points after stimulation with different Th1- and Th2-polarizing pathogen-derived molecules. The samples were measured using liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for relative quantitation. Results: Lipopolysaccharide-induced maturation promoted the expression of proteins related to metabolic, cellular, and immune system processes. Th1-polarizing DCs, conditioned by IFN-γ during maturation, displayed accelerated maturation by differentially expressing cytoskeletal proteins and proteins involved in immune regulation. The stimulation of DCs with soluble egg antigens and omega-1 derived from Schistosoma mansoni, which are both Th2-inducing stimuli, increased 60S acidic ribosomal protein P2, and vesicle amine transferase 1 while decreasing the expression of proteins related to antigen processing and presentation. Conclusion: Our data indicate that not only proteins involved in the interaction between T cells and DCs at the level of the immunological synapse, but also those related to cellular metabolism and stress, may promote Th2 polarization.

Wolbachia Modulates Lipid Metabolism in Aedes albopictus Mosquito Cells.

ABSTRACTCertain strains of the intracellular endosymbiont Wolbachia can strongly inhibit or block the transmission of viruses such as dengue virus (DENV) by Aedes mosquitoes, and the mechanisms responsible are still not well understood. Direct infusion and liquid chromatography-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry-based lipidomics analyses were conducted using Aedes albopictus Aa23 cells that were infected with the wMel and wMelPop strains of Wolbachia in comparison to uninfected Aa23-T cells. Substantial shifts in the cellular lipid profile were apparent in the presence of Wolbachia. Most significantly, almost all sphingolipid classes were depleted, and some reductions in diacylglycerols and phosphatidylcholines were also observed. These lipid classes have previously been shown to be selectively enriched in DENV-infected mosquito cells, suggesting that Wolbachia may produce a cellular lipid environment that is antagonistic to viral replication. The data improve our understanding of the intracellular interactions between Wolbachia and mosquitoes.IMPORTANCE Mosquitoes transmit a variety of important viruses to humans, such as dengue virus and Zika virus. Certain strains of the intracellular bacterial genus called Wolbachia found in or introduced into mosquitoes can block the transmission of viruses, including dengue virus, but the mechanisms responsible are not well understood. We found substantial shifts in the cellular lipid profiles in the presence of these bacteria. Some lipid classes previously shown to be enriched in dengue virus-infected mosquito cells were depleted in the presence of Wolbachia, suggesting that Wolbachia may produce a cellular lipid environment that inhibits mosquito-borne viruses.

Boosting Sensitivity in Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance-Tandem Mass Spectrometry for Product Ion Analysis of Monoterpene Indole Alkaloids.

In metabolomics, the analysis of product ions in tandem mass spectrometry (MS/MS) is noteworthy to chemically assign structural information. However, the development of relevant analytical methods are less advanced. Here, we developed a method to boost sensitivity in liquid chromatography–Fourier transform ion cyclotron resonance–tandem mass spectrometry analysis (MS/MS boost analysis). To verify the MS/MS boost analysis, both quercetin and uniformly labeled 13C quercetin were analyzed, revealing that the origin of the product ions is not the instrument, but the analyzed compounds resulting in sensitive product ions. Next, we applied this method to the analysis of monoterpene indole alkaloids (MIAs). The comparative analyses of MIAs having indole basic skeleton (ajmalicine, catharanthine, hirsuteine, and hirsutine) and oxindole skeleton (formosanine, isoformosanine, pteropodine, isopteropodine, rhynchophylline, isorhynchophylline, and mitraphylline) identified 86 and 73 common monoisotopic ions, respectively. The comparative analyses of the three pairs of stereoisomers showed more than 170 common monoisotopic ions in each pair. This method was also applied to the targeted analysis of MIAs in Catharanthus roseus and Uncaria rhynchophylla to profile indole and oxindole compounds using the product ions. This analysis is suitable for chemically assigning features of the metabolite groups, which contributes to targeted metabolome analysis.

5-Diethylamino-naphthalene-1-sulfonyl Chloride (DensCl): A Novel Triplex Isotope Labeling Reagent for Quantitative Metabolome Analysis by Liquid Chromatography Mass Spectrometry

We describe a new set of isotope reagents, (12)C4-, (12)C2(13)C2-, and (13)C4-5-diethylamino-naphthalene-1-sulfonyl chloride (DensCl), in combination with liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), for improved analysis of the amine- and phenol-containing submetabolome. The synthesis of the reagents is reported, and an optimized derivatization protocol for labeling amines and phenols is described. To demonstrate the utility of the triplex reagents for metabolome profiling of biological samples, urine samples collected daily from a healthy volunteer over a period of 14 days were analyzed. The overall workflow is straightforward, including differential isotope labeling of individual samples and a pooled sample that serves a global internal standard, mixing of the isotope differentially labeled samples and LC-MS analysis for relative metabolome quantification. Comparing to the dansyl chloride (DnsCl) duplex isotope reagents, the new triplex DensCl reagents offer the advantages of improved metabolite detectability due to enhanced sensitivity (i.e., about 1000 peak pairs detected by DensCl labeling vs about 600 peak pairs detected by DnsCl labeling) and analysis speed (i.e., simultaneous analysis of two comparative samples by DensCl vs only one comparative sample analyzed by DnsCl).

Liquid–liquid extraction combined with differential isotope dimethylaminophenacyl labeling for improved metabolomic profiling of organic acids

A large fraction of the known human metabolome belong to organic acids. However, comprehensive profiling of the organic acid sub-metabolome is a major analytical challenge. In this work, we report an improved method for detecting organic acid metabolites. This method is based on the use of liquid–liquid extraction (LLE) to selectively extract the organic acids, followed by using differential isotope p-dimethylaminophenacyl (DmPA) labeling of the acid metabolites. The 12C-/13C-labeled samples are analyzed by liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC–FTICR–MS). It is shown that this LLE DmPA labeling method offers superior performance over the method of direct DmPA labeling of biofluids such as human urine. LLE of organic acids reduces the interference of amine-containing metabolites that may also react with DmPA. It can also remove water in a biofluid that can reduce the labeling efficiency. Using human urine as an example, it is demonstrated that about 2500 peak pairs or putative metabolites could be detected in a 30-min gradient LC–MS run, which is about 3 times more than that detected in a sample prepared using direct DmPA labeling. About 95% of the 1000 or so matched metabolites to the Human Metabolome Database (HMDB) are organic acids. It is further shown that this method can be used to handle as small as 10μL of urine. We believe that this method opens the possibility of generating a very comprehensive profile of the organic acid sub-metabolome that will be useful for comparative metabolomics applications for biological studies and disease biomarker discovery.

Combination of Liquid Chromatography–Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry with 13C-Labeling for Chemical Assignment of Sulfur-Containing Metabolites in Onion Bulbs

Phytochemicals containing heteroatoms (N, O, S, and halogens) often have biological activities that are beneficial to humans. Although targeted profiling methods for such phytochemicals are expected to contribute to rapid chemical assignments, thus making phytochemical genomics and crop breeding much more efficient, there are few profiling methods for the metabolites. Here, as an ultrahigh performance approach, we propose a practical profiling method for S-containing metabolites (S-omics) using onions (Allium cepa) as a representative species and (12)C- and (13)C-based mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses by liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry (LC-FTICR-MS). Use of the ultrahigh quality data from FTICR-MS enabled simplifying the previous methods to determine specific elemental compositions. MS analysis with a resolution of >250,000 full width at half-maximum and a mass accuracy of <1 ppm can distinguish S-containing monoisotopic ions from other ions on the basis of the natural abundance of (32)S and (34)S and the mass differences among the S isotopes. Comprehensive peak picking using the theoretical mass difference (1.99579 Da) between (32)S-containing monoisotopic ions and their (34)S-substituted counterparts led to the assignment of 67 S-containing monoisotopic ions from the (12)C-based MS spectra, which contained 4693 chromatographic ions. The unambiguous elemental composition of 22 ions was identified through comparative analysis of the (12)C- and (13)C-based MS spectra. Finally, of these, six ions were found to be derived from S-alk(en)ylcysteine sulfoxides and glutathione derivatives. This S-atom-driven approach afforded an efficient chemical assignment of S-containing metabolites, suggesting its potential application for screening not only S but also other heteroatom-containing metabolites in MS-based metabolomics.

Natural hypolignification is associated with extensive oligolignol accumulation in flax stems.

Flax (Linum usitatissimum) stems contain cells showing contrasting cell wall structure: lignified in inner stem xylem tissue and hypolignified in outer stem bast fibers. We hypothesized that stem hypolignification should be associated with extensive phenolic accumulation and used metabolomics and transcriptomics to characterize these two tissues. (1)H nuclear magnetic resonance clearly distinguished inner and outer stem tissues and identified different primary and secondary metabolites, including coniferin and p-coumaryl alcohol glucoside. Ultrahigh-performance liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry aromatic profiling (lignomics) identified 81 phenolic compounds, of which 65 were identified, to our knowledge, for the first time in flax and 11 for the first time in higher plants. Both aglycone forms and glycosides of monolignols, lignin oligomers, and (neo)lignans were identified in both inner and outer stem tissues, with a preponderance of glycosides in the hypolignified outer stem, indicating the existence of a complex monolignol metabolism. The presence of coniferin-containing secondary metabolites suggested that coniferyl alcohol, in addition to being used in lignin and (neo)lignan formation, was also utilized in a third, partially uncharacterized metabolic pathway. Hypolignification of bast fibers in outer stem tissues was correlated with the low transcript abundance of monolignol biosynthetic genes, laccase genes, and certain peroxidase genes, suggesting that flax hypolignification is transcriptionally regulated. Transcripts of the key lignan genes Pinoresinol-Lariciresinol Reductase and Phenylcoumaran Benzylic Ether Reductase were also highly abundant in flax inner stem tissues. Expression profiling allowed the identification of NAC (NAM, ATAF1/2, CUC2) and MYB transcription factors that are likely involved in regulating both monolignol production and polymerization as well as (neo)lignan production.

Clinical perspectives of high-resolution mass spectrometry–based proteomics in neuroscience—Exemplified in amyotrophic lateral sclerosis biomarker discovery research

Biomarker discovery is a central application in today's proteomic research. There is an urgent need for valid biomarkers to improve diagnostic tools and treatment in many disorders, such as the rapidly progressing neurodegenerative disorder amyotrophic lateral sclerosis (ALS) that has a fatal outcome in about 3 years and yet no curative treatment. Screening for clinically relevant biomarkers puts high demands on high-throughput, rapid and precise proteomic techniques. There is a large variety in the methods of choice involving mainly gel-based approaches as well as chromatographic techniques for multi-dimensional protein and peptide separations followed by mass spectrometry (MS) analysis. This special feature article will discuss some important aspects of MS-based clinical proteomics and biomarker discovery in the field of neurodegenerative diseases and ALS research respectively, with the aim to provide a prospective view on current and future research aspects in the field. Furthermore, examples for application of high-resolution MS-based proteomic strategies for ALS biomarker discovery will be demonstrated with two studies previously reported by our group. These studies include among others, utilization of capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) for advanced protein pattern classification in cerebrospinal fluid (CSF) samples of ALS patients as well as highly sensitive protein identification in minimal amounts of postmortem spinal cord tissue and laser micro-dissected motor neurons using FT-ICR-MS in conjunction with nanoflow LC coupled to matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (LC-MALDI-TOF-TOF-MS).

Steady-State Asymmetric Nanospray Dual Ion Source for Accurate Mass Determination within a Chromatographic Separation

Here we report the design, implementation, and initial use of an asymmetric steady-state continuous dual-nanospray ion source. This new source design consists of two independently controlled and continuously operating nanospray interfaces with funnel shaped counter electrodes. A steady-state ion mixing region combines the ions from the two sources into a single ion beam in the intermediate region after ion extraction from the nanospray sources but before the bulk of the pressure gradient of the vacuum interface. With this design we have achieved robust mixing of ions with no loss of duty cycle and remarkable ionization characteristics that appear entirely noncompetitive and potentially beneficial. This allows continuous introduction of internal mass calibration ions during a liquid chromatography-mass spectrometric analysis. This in turn allows for recalibration of individual spectra yielding sub part per million mass accuracy throughout the run. The steady-state approach presented here has several advantages over previous approaches. Since neither the voltage nor positions of the sprayers are changed, the nanospray has greater spray stability. The ions produced by the analyte sprayer are continuously sampled, as opposed to time-sharing which necessitates that the analyte ion stream be interrupted for some part of the duty cycle. There are no moving parts, no rapid changes to high voltages requiring additional control electronics, and no need for completely separate vacuum interfaces and the associated complexity. The sprayers are independently controlled and do not exhibit competition or mutual ionization suppression. This novel source has been implemented with a Bruker Apex II 9.4 T FTICR with a modified Apollo electrospray ion source as part of a nanoflow liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry analysis platform. Because of the low cost of implementation, the new source could potentially be applied to other forms of mass spectrometry, such as electrospray ionization-time-of-flight (ESI-TOF), which can benefit from internal mass calibration.

Analysis of Ginsenosides in Ginseng Drugs Using Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Analysis of ginsenosides in five Ginseng drugs derived from Panax ginseng (white ginseng), P. quinquefolius, P. japonicus produced in Japan, P. notoginseng, and P. vietnamensis using Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (LC-FTICR-MS) was performed. Ginsenosides in the drugs were identified by the molecular formula obtained from high-resolution mass data and multiple stage MS/MS analysis. Twenty-six known ginsenosides were identified as the major constituents in the extracts of the Ginseng drugs. The five Ginseng drugs showed different reconstructed mass chromatographic profiles and were discriminated from each other.

Normalization Approaches for Removing Systematic Biases Associated with Mass Spectrometry and Label-Free Proteomics

Central tendency, linear regression, locally weighted regression, and quantile techniques were investigated for normalization of peptide abundance measurements obtained from high-throughput liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS). Arbitrary abundances of peptides were obtained from three sample sets, including a standard protein sample, two Deinococcus radiodurans samples taken from different growth phases, and two mouse striatum samples from control and methamphetamine-stressed mice (strain C57BL/6). The selected normalization techniques were evaluated in both the absence and presence of biological variability by estimating extraneous variability prior to and following normalization. Prior to normalization, replicate runs from each sample set were observed to be statistically different, while following normalization replicate runs were no longer statistically different. Although all techniques reduced systematic bias to some degree, assigned ranks among the techniques revealed that for most LC-FTICR-MS analyses linear regression normalization ranked either first or second. However, the lack of a definitive trend among the techniques suggested the need for additional investigation into adapting normalization approaches for label-free proteomics. Nevertheless, this study serves as an important step for evaluating approaches that address systematic biases related to relative quantification and label-free proteomics.

Cerebrospinal fluid protein patterns in neurodegenerative disease revealed by liquid chromatography‐Fourier transform ion cyclotron resonance mass spectrometry

This study demonstrates the power of a novel proteomic approach developed for the detection and identification of biological markers in body fluids. The goal was to observe alterations in the protein patterns of cerebrospinal fluid (CSF) related to amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder with unknown etiology. In the experiments, tryptic digests of CSF from patients and healthy controls were analyzed by on-line capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry. (FT-ICR MS) Typically, around 4000 peptides were detected in one such experiment, and a pattern recognition program was constructed for the data analysis to distinguish mass chromatograms from patients and controls. This strategy was evaluated comparing the peptide patterns of CSF spiked in vitro with a biomarker, with control CSF. The patterns were clearly separated and the tryptic peptides of the biomarker were successfully selected as characteristic peaks. Hence, the method was applied to compare mass chromatograms of CSF from 12 ALS-patients and 10 matched healthy controls. While no biomarker alone could be identified from the characteristic peaks, we were able to assign 4 out of 5 unknown samples correctly (i.e., 80% correctly diagnosed, 20% false-negative), and it would be 100% if we reject a possible outlier believed to be caused by an occlusion in the spinal CSF compartment. These findings are very promising, although the clinical relevance is not fully established due to the low number of unknown samples analyzed. In addition to the diagnostic potential, these results may be important steps towards understanding the neurodegenerative process.

An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics

We describe a fully automated high performance liquid chromatography 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer system designed for proteomics research. A synergistic suite of ion introduction and manipulation technologies were developed and integrated as a high-performance front-end to a commercial Bruker Daltonics FTICR instrument. The developments incorporated included a dual-ESI-emitter ion source; a dual-channel electrodynamic ion funnel; tandem quadrupoles for collisional cooling and focusing, ion selection, and ion accumulation, and served to significantly improve the sensitivity, dynamic range, and mass measurement accuracy of the mass spectrometer. In addition, a novel technique for accumulating ions in the ICR cell was developed that improved both resolution and mass measurement accuracy. A new calibration methodology is also described where calibrant ions are introduced and controlled via a separate channel of the dual-channel ion funnel, allowing calibrant species to be introduced to sample spectra on a real-time basis, if needed. We also report on overall instrument automation developments that facilitate high-throughput and unattended operation. These included an automated version of the previously reported very high resolution, high pressure reversed phase gradient capillary liquid chromatography (LC) system as the separations component. A commercial autosampler was integrated to facilitate 24 h/day operation. Unattended operation of the instrument revealed exceptional overall performance: Reproducibility (1–5% deviation in uncorrected elution times), repeatability (<20% deviation in detected abundances for more abundant peptides from the same aliquot analyzed a few weeks apart), and robustness (high-throughput operation for 5 months without significant downtime). When combined with modulated-ion-energy gated trapping, the dynamic calibration of FTICR mass spectra provided decreased mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations over a dynamic range of peptide peak intensities for each spectrum of 10 3, and >10 5 for peptide abundances in the overall separation.

Liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometric characterization of protein kinase C phosphorylation.

A vented column, capillary liquid chromatography (LC) microelectrospray ionization (ESI) Fourier transform ion cyclotron resonance (FT-ICR (9.4 T)) mass spectrometry (MS) approach to phosphopeptide identification is described. A dual-ESI source capable of rapid (approximately 200 ms) switching between two independently controlled ESI emitters was constructed. The dual-ESI source, combined with external ion accumulation in a linear octopole ion trap, allowed for internal calibration of every mass spectrum during LC. LC ESI FT-ICR positive-ion MS of protein kinase C (PKC) revealed four previously unidentified phosphorylated peptides (one within PKC(alpha), one within PKC(delta), and two within PKC(zeta)). Internal calibration improved the mass accuracy for LC MS spectra from an absolute mean (47 peptide ions) of 11.5 ppm to 1.5 ppm. Five additional (out of eight known) activating sites of PKC phosphorylation, not detected in positive-ion experiments, were observed by subsequent negative-ion direct infusion nanoelectrospray. Extension of the method to enable infrared multiphoton dissociation of all ions in the ICR cell prior to every other mass measurement revealed the diagnostic neutral loss of H3PO4 from phosphorylated peptide ions. The combination of accurate-mass MS and MS/MS offers a powerful new tool for identifying the presence and site(s) of phosphorylation in peptides, without the need for additional wet chemical derivatization.

{BLR 400} Legislation - U.S. Congress - Patent - Regulation

Biotechnology Law ReportVol. 4, No. 7-8 {BLR 400} Legislation - U.S. Congress - Patent - RegulationPATENT TERM RESTORATION BILL INTRODUCED IN SENATE —Legislation Would Restore Patent Term for Agrichemical ProductsPublished Online:23 Mar 2009https://doi.org/10.1089/blr.1985.4.235AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byAn automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics1 February 2004 | Journal of the American Society for Mass Spectrometry, Vol. 15, No. 2Electrospray ionization-Fourier transform ion cyclotron mass spectrometry using ion preselection and external accumulation for ultrahigh sensitivity1 January 2001 | Journal of the American Society for Mass Spectrometry, Vol. 12, No. 1A dual vacuum chamber Fourier transform mass spectrometer with rapidly interchangeable FAB, MALDI and ESI sources: electrospray resultsInternational Journal of Mass Spectrometry and Ion Processes, Vol. 157-158Possible applications of an external resonant circuit in Fourier transform ion cyclotron resonanceRapid Communications in Mass Spectrometry, Vol. 9, No. 4External ion source FTMS instrument for analysis of high mass ionsInternational Journal of Mass Spectrometry and Ion Processes, Vol. 87, No. 1 Volume 4Issue 7-8Jul 1985 To cite this article:{BLR 400} Legislation - U.S. Congress - Patent - Regulation.Biotechnology Law Report.Jul 1985.235-271.http://doi.org/10.1089/blr.1985.4.235Published in Volume: 4 Issue 7-8: March 23, 2009PDF download