High Resolution Selected Ion Monitoring Research Articles

Quantitative Proteomics Identify Urinary Hgfl Protein As a Potential Marker for the Development of Chronic Kidney Disease in Sickle Cell Disease Patients

BACKGROUND: Chronic kidney disease (CKD) is common in patients with sickle cell disease (SCD). However, the progression of CKD in SCD and factors associated with such progression remain poorly defined. Liquid chromatography mass spectrometry (LC-MS) based quantitative proteomics has become a highly potent method for biomarker discovery due to growing capabilities for broad proteome coverage and good accuracy and precision in quantification.OBJECTIVES: The purpose of this study was to identify the potential markers associated with CKD progression in patients with SCD using quantitative proteomics.METHODS: Urine samples were collected from healthy controls and SCD patients with different CKD stages from University of Illinois at Chicago (UIC). Mass-spectrometry analysis was performed on an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) coupled to a Prominence Nano LC (Shimadzu) using the Xcalibur version 2.7.0 (Thermo Scientific). Proteome Discoverer 1.4 and SIEVE 2.1 programs were used for protein identification and label-free quantification. Heavy isotope labeled peptide EDQTSPAPGLR(13C6, 15N) was used as an internal standard for high resolution/selected ion monitoring (HR/SIM) analysis of HGFL. Urinary HGFL protein together wiht creatinine and albumin were also measured by ELISA.RESULTS: Since glomerular hyperfiltration is an early stage of renal dysfunction. We performed label-free quantitative proteomic analysis for urine samples collected from SCD patients with hyperfiltration (N=3) and normal (N=3). Hepatocyte growth factor-like (HGFL) protein was found to be significantly downregulated (5.52-fold, p=8.05 × 10-5) in samples with glomerular hyperfiltration compared to normal group. Next, we developed a high resolution/selected ion monitoring (HR/SIM) method by measuring the HGFL peptide (m/z 585.79) with isotope labeled-HGFL peptide (m/z 590.80) as internal standard (IS). HR/SIM quantification was performed for 19 urine samples from SCD patients and 12 urine samples from healthy controls. HGFL levels were found to be significantly downregulated (p=0.0084) in the SCD urine samples compared to samples from healthy controls (Figure 1). To further assess the correlation between HGFL level and CDK stage, we expanded the analysis to SCD patients with different CKD stage ranging from 0 to 5 and 19 healthy individuals by ELISA. The result confirmed the finding of HR/SIM quantification, moreover, showed that urinary HGFL level highly correlated with CKD stage (r= ̶ 0.4106, p=0.002, Figure 1) and showed high sensitivity and specificity by Receiver Operating Characteristic (ROC) curve analysis (AUC=0.78).CONCLUSIONS: HGFL protein has been identified as a negative regulator of phosphatidylinositol 3-kinase (PI3K), and PI3K/Akt pathway was found to be activated in the progress of CKD. Therefore, the decrease of HGFL level in urines from SCD patients may indicate the development of CKD. Combination of LC-MS based quantitative proteomics and ELISA validation is an useful approach for biomarker discovery.ACKNOWLEDGMENTS: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005, 5G12MD007597 and K23HL125984. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. [Display omitted] DisclosuresGordeuk:Emmaus Life Sciences: Consultancy.

Elevated Levels of Hgfl Protein in Sickle Cell Disease Urine Samples That Induce Glomerular Permeability

Background: Chronic kidney disease (CKD) is a prevalent complication of sickle cell disease (SCD) associated with early mortality. Hemoglobinuria is a risk factor for the development of albuminuria and CKD. Currently, there are no biomarkers that predict outcome of CKD. Mass-spectrometry analysis of patient urine is a highly potent modern method for biomarker discovery. An in vitro glomerular permeability assay has been used as a non-invasive test for glomerular disease prognosis. Application of this assay to urinary samples collected before kidney disease onset provides a unique opportunity for differential proteomics of a limited number of urinary proteins. In combination with high resolution/selected ion monitoring (HR/SIM) mass-spectrometry methodology, this experimental platform provides an opportunity for biomarker discovery and validation of proteins whose concentration is too low to be detected by an immunological assay.Objectives: We aimed to determine a biomarker of early stage kidney disease using samples collected from the patients of the Center for Sickle Cell Disease at Howard University. We then used HR/SIM method to validate this biomarker in the cohort of SCD patients with and without CKD from University of Illinois at Chicago (UIC).Methods:Urinary protein, creatinine, albumin were measured by ELISA. The pH and specific gravity (SG) were determined by Multistix. Glomeruli were isolated from the murine kidney (FVB/N strain) and albumin permeability (Palb) activity was determined using urine samples collected from patients of the Center for Sickle Cell Disease, Howard University. Mass-spectrometry analysis was performed and Protein Discovery 1.4 and SIEVE 2.0 programs were used for protein analysis and label-free quantification. Heavy isotope labeled peptide EDQTSPAPGLR(13C6, 15N) was used as an internal standard for HR/SIM analysis of the samples from UIC.Results: Glomerular permeability assay was performed using six urinary samples collected from patients of the Center for Sickle Cell Disease, Howard University. Mass-spectrometry assay was performed for all samples. Samples with similar values of protein, albumin, creatinine, pH and SG, but different Palb activity were compared by SIEVE 2.0. Higher levels of hepatocyte growth factor-like (HGFL) protein were observed in three samples that induced glomerular permeability compared to three samples that did not. Since our attempts to produce antibodies to HGFL peptide for an ELISA assay were unsuccessful, we developed a HR/SIM method to measure HGFL peptide in urine. HR/SIM was performed for eight urine samples from the UIC cohort by measuring the ratio of ion peaks of HGFL peptide (m/z 585.79) and internal standard (IS) (m/z 590.80) (Fig. 1A). HGFL levels were found to be significantly increased (1.72-fold, p=0.015) in the urine samples of two patients at risk of developing renal disease based on the presence of hemoglobinuria compared to six samples without hemoglobinuria (Fig. 1B).Conclusions: Combination of in vitro glomerular permeability assay with mass-spectrometry method of protein discovery and HR/SIM validation assay may be a useful platform for discovery of biomarker of early stage of CKD. Urinary level of HGFL may serve as a prognostic marker for development of CKD in SCD patients. A limitation of our study is the small number of samples used for validation.Acknowledgments: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Absolute Quantification of a Steroid Hormone that Regulates Development in Caenorhabditis elegans

Under favorable conditions, Caenorhabditis elegans larvae grow into reproductive adults after a series of molting cycles. When environmental conditions are harsh, they arrest as dauer larvae. Dafachronic acid (DA), a C. elegans steroid hormone, is required for reproductive development. Here, we report a mass spectrometry (MS) method for absolute quantitation of DA in C. elegans. The extraction of DA from C. elegans was optimized to achieve a recovery rate of greater than 83%. The MS sensitivity to DA increased 100-fold after carboxyl group derivatization with 2-picolylamine. High-resolution selected ion monitoring (HR-SIM) on a Q-Orbitrap mass spectrometer Q Exactive outperformed targeted-MS2 on the same instrument and selected reaction monitoring (SRM) on a triple-quadrupole mass spectrometer TSQ Quantum Discovery. With a limit of quantification as low as 1 pg of DA, the HR-SIM method enables absolute quantification of endogenous DA during the reproductive development of C. elegans. We found that in wild-type (WT) worms, DA increases from 0.04 ± 0.02 ng/mg protein in the L1 larval stage to 1.21 ± 0.67 ng/mg protein in the L2 larval stage and decreases again after the L3 stage. In comparison, four genetic mutants that have a constitutive dauer-formation phenotype due to disrupted insulin, TGF-β, or cGMP signaling all have a very low DA level in the L2 stage (below 15% of the WT). These mutants are able to escape the dauer fate and most of them grow into fertile adults when supplied with exogenous DA. Therefore, a DA spike in the L2 stage is critical for the reproductive development of C. elegans.

Quantifying Peptides in Complex Mixtures with High Sensitivity and Precision Using Targeted Approach with a Hybrid Linear Ion Trap Orbitrap Mass Spectrometer

High‐resolution, accurate‐mass (HR/AM) mass spectrometry is routinely used for identification and relative quantitation of peptides present in complex mixtures using stable‐isotope labeling, isobaric tags, or label‐free techniques. However, HR/AM application for targeted peptide analysis is less explored. Due to the complexity of biological samples, accurate mass and retention time alone are not sufficient for verifying identity of a targeted peptide. Therefore, sequence confirmation via MS/MS is essential during the early verification and validation stages of development of a targeted quantitative assay. Here, a workflow was developed for targeted protein/peptide quantitation on the LTQ Orbitrap Velos to use high resolution selected ion monitoring (SIM) for quantitation followed by targeted MS/MS for sequence confirmation. A standard peptide mixture spiked into a yeast digest mixture at different concentration as quantitative targets and LOD was as low as 10 amol. An additional yeast digest was used to further evaluate the precision and throughput and the workflow by 77 peptides (25 yeast proteins). All the peptides were successfully quantified and verified with high analytical precision in a single LC/MS run.

The dissipation of tebuconazole and propiconazole in boronia (Boronia megastigma Nees).

The broad spectrum, systemic fungicides tebuconazole and propiconazole are used to control rust in boronia (Boronia megastigma Nees). Gas chromatography combined with either a benchtop quadrupole mass spectrometer or a high-resolution mass spectrometer allowed for the monitoring of both pesticides in boronia leaves, flowers, and concrete. Field trials were established at two sites to determine the rate of dissipation of tebuconazole and propiconazole in boronia. At site 1, two application rates of 125 and 250 g active ingredient/hectare (ai/ha) tebuconazole were employed. Treatments were repeated 17 days later. At harvest, 286 days after the final application, tebuconazole was detected at levels of 0.06 +/- 0.05 and 0.5 +/- 0.1 [mg/kg +/- standard error, on a dry matter basis (DMB)] in the leaves collected from plots treated with 125 and 250 g ai/ha of tebuconazole, respectively. The oil produced from the flowers collected at the final harvest had residues of tebuconazole at levels of 0.06 +/- 0.03 and 0.10 +/- 0.08 mg/kg for the 125 and 250 g ai/ha application rates, respectively. Two repeat applications of 125 g ai/ha propiconazole were also used at site 1. Residues of propiconazole were detected at 0.09 +/- 0.03 mg/kg (DMB) 286 days after the final application. At site 2, treatments of 125 g ai/ha of tebuconazole were applied twice. At harvest, 279 days after the final application of tebuconazole, residues were recorded at 0.30 +/- 0.09 mg/kg in the leaves (DMB) while the oil produced had 0.20 +/- 0.07 mg/kg.

Mass spectrometric characterization of Q1, a C(9)H(3)Cl(7)N(2) contaminant in environmental samples

Q1, a heptachloro component of unknown structure and origin, was recently identified as a major organochlorine contaminant in samples from Africa and the Antarctic. Gas chromatography in combination with low resolution mass spectrometry (LRMS) was applied to establish a molecular weight of m/z 384 including seven chlorine atoms. Three possible molecular formulae (C(11)H(7)Cl(7), C(10)H(3)Cl(7)O, and C(9)H(3)Cl(7)N(2)) were proposed which could not be distinguished by LRMS. In this presentation the molecular composition of Q1 was studied using gas chromatography in combination with high resolution electron impact ionization mass spectrometry. With the option of further heteroatoms (P, S, N, O, F, and Br), 17 molecular formulae were obtained for the molecular weight of 384 u. In the selected ion monitoring (SIM) mode, performed with a resolution of 16,000, highest response was found at 383.812 or C(9)H(3)Cl(7)N(2). 11 fragment ions detected in the low resolution full scan mass spectrum of Q1 were also investigated in the high resolution SIM mode. In every case, the nitrogen-variant showed highest abundance while the other 16 structural variants could be definitely excluded. These investigations revealed that the molecular formula of Q1 is C(9)H(3)Cl(7)N(2). No stable component with this molecular formula has ever been reported in the literature, to our knowledge. Copyright 1999 John Wiley & Sons, Ltd.

Analysis of ascorbate and dehydroascorbate in plant extracts by high-resolution selected ion monitoring gas chromatography-mass spectrometry

Here we report on the quantitative analysis of tert.-butyldimethylsilyl derivatives, of ascorbate and dehydroascorbate in plant extracts by high-resolution selected ion monitoring gas chromatography-mass spectrometry. The analysis involved an isotope dilution assay using [ 13C 1]ascorbate and [ 13C 1]dehydroascorbate. The limit of detection was approximately 0.5 pg for ascorbate and 5 pg for dehydroascorbate. The reproductibility of the whole quantification procedure, from extraction of Pinus sylvestris needles with 5% trichloroacetic acid to the final analysis, was very high: giving a relative standard deviation (R.S.D.) of 4% and 10% for ascorbate and dehydroascorbate, respectively. The reproducibility of the analysis of the redox ratio of ascorbate/(ascorbate + dehydroascorbate) was even higher (R.S.D. = 1%). The high-resolution selected ion monitoring gas chromatography-mass spectrometry methodology developed here with very high sensitivity and specificity for ascorbate and dehydroascorbate opens new perspectives for investigations on distribution patterns of these substances in plants.

High-Resolution Selected Ion Monitoring in a Quadrupole Ion Trap Mass Spectrometer

A new method of selected ion monitoring was described that was capable of unit mass isolation throughout the operational mass range of an ion trap mass spectrometer. The trap was first selectively filled with ions via the application of a broad-band waveform to the end caps during ionization. A second isolation step completed the unit mass isolation, leaving only the desired ions in the trap. Following the accumulation and isolation steps, the intensity of the isolated ions was determined by resonantly scanning them from the trap for detection. As many as 10 different masses could be monitored by sequentially applying these steps. The ionization time for each mass was individually adjusted to optimize the filling of the trap with the selected ions ; this increased the dynamic range for the set of ions. The individual spectra for each mass could then be added together to form a mass spectrum containing all specified ions.

Comparison between high-resolution selected ion monitoring, selected reaction monitoring, and four-sector tandem mass spectrometry in quantitative analysis of gibberellins in milligram amounts of plant tissue.

Comparison between high-resolution selected ion monitoring, selected reaction monitoring, and four-sector tandem mass spectrometry in quantitative analysis of gibberellins in milligram amounts of plant tissue.

Determination of Propiconazole Residue in Boronia Extract (Concrete)

A method for detecting residues of the fungicide propiconazole in the petroleum ether extract (concrete) of Boronia megastigma Nees was developed. Gas chromatography/mass spectrometry (GC/MS) using low-resolution selected ion monitoring (LR SIM) had a 1 ppm detection limit in samples that had some preliminary cleanup. The use of GC/MS with high-resolution selected ion monitoring (HR SIM) had a minimum detection limit of 50 ppb for crude spiked samples of boronia concrete. This method was utilized to provide an automated assay for propiconazole residues in the concrete from boronia flowers and in extracts of vegetative material. © 1995 American Chemical Society.

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography‐mass spectrometry. GA1 GA19 GA20 and GA29 were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA1. GA19. GA20 and putative GA29 and GA53 were quantified in the apical buds, while GA4. GA8. GA9 and GA44 were shown to be either absent or present at very low levels. From the cambial region. GA1 and GA20 were quantified at levels of 0.30 ng (g fresh weight)‐1 and 8.8 ng (g fresh weight)‐1 respectively. These data suggest that the early 13‐hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus.

Quantitative determination of F6-1,25(OH)2 vitamin D3 in human serum by gas chromatography/mass spectrometry with high-resolution selected ion monitoring

A highly sensitive and specific gas chromatographic/mass spectrometric method was developed for the determination of very low levels of F6-1,25(OH)2 vitamin D3, a new fluoro analogue of vitamin D3, to facilitate investigation of its pharmacokinetics in man. Deuterium-labelled F6-1,25(OH)2 vitamin D3 was employed as an internal standard for quantification. The method involves extraction from serum samples, separation by a cartridge column system followed by straight-phase high-performance liquid chromatography, O-trimethylsilylation, and analysis by gas chromatography/mass spectrometry using high-resolution selected ion monitoring. With a quantification limit of 2 pg ml-1 and a requirement for 2 ml of serum, the assay method has sufficient sensitivity, selectivity and precision for the analysis of very low concentrations of the drug.

Simultaneous determination of mevalonate and 7α-hydroxycholesterol in human plasma by gas chromatography—mass spectrometry as indices of cholesterol and bile acid biosynthesis

A very sensitive and specific method for the simultaneous determination of mevalonate and 7α-hydroxycholesterol in human plasma is described. The assay is based on isotope dilution mass spectrometry: the extracts from plasma were treated with benzylamine followed by dimethylethylsilylimidazole, then the resulting dimethylethylsilyl ether derivatives of mevalonylbenzylamide and 7α-hydroxycholesterol were determined by gas chromatography—mass spectrometry using high-resolution selected-ion monitoring. Simple regression analysis revealed significant correlations between the plasma level of mevalonate and the hepatic activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34) ( r = 0.83, P < 0.01) and between the plasma level of free 7α-hydroxycholesterol and the hepatic activity of cholesterol 7α-hydroxylase (EC 1.14.13.7) ( r = 0.76, P < 0.05).

Simultaneous determination of testosterone and androstadienone (sex attractant) in human plasma by gas chromatography—mass spectrometry with high-resolution selected-ion monitoring

Androsta-4,16-dien-3-one (androstadienone) and androst-4-en-3-one-17β-ol (testosterone) in healthy human plasma were simultaneously determined under several experimental conditions by gas chromatography—mass spectrometry with high-resolution selected-ion monitoring. Internal standards were [2,2,4,6,6- 2H 5]androstadienone and [2,2,4,6,6- 2H 5]testosterone. Samples were extracted with an Extrelut ® column, purified using Lipidex ® 5000 and converted into hydroxime-trimethylsilyl derivatives for determination. Physiological concentrations of androstadienone and testosterone found in eleven healthy men were 2.05 ± 0.74 and 18.6 ± 4.9 pmol/ml in plasma (mean ± S.D.), respectively. No correlation was observed between these steroid concentrations.

Analysis of rat brain microdialysate by gas chromatography—high-resolution selected-ion monitoring mass spectrometry

Gas chromatography—high-resolution selected-ion monitoring mass spectrometry was used to analyze catecholamine metabolites in rat brain microdialysate. Dialysate samples were collected in vials containing stable isotope analogues of homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylglycol (MHPG) and 5-hydroxyindoleacetic acid (5HIAA) and analyzed as their trimethylsilyl derivatives. The metabolite levels were monitored at 20-min intervals throughout the time course of the experiment, beginning immediately after surgery and implantation of the dialysis probe and ending 4 h after amphetamine treatment. The levels of HVA were observed to decrease after amphetamine treatment, while those of MHPG and 5HIAA did not change significantly.

Quantitative analysis of hexythiazox by direct insertion probe mass specytrometry

A quantitative direct insertion probe/mass spectrometry (DIP/MS) method is described for the analysis of the miticide hexythiazox. Fruit (50 g) was extracted by blending with ethyl acetate (100 ml) and the crude ethyl acetate extract analysed by DIP/MS using the three signal monitoring methods: low-resolution selected ion monitoring (SIM), high-resolution SIM and selected decomposition monitoring (SDM). Detection limits achieved were 0.1, 0.1 and 0.02 mg kg−1 for low-resolution SIM, high-resolution SIM and SDM, respectively. DIP/SDM/MS was the preferred monitoring method as it was straightforward to use and, as SDM is a more selective monitoring method, a lower detection limit was achieved when crude fruit extracts were analysed.

High resolution selected ion monitoring of 5-methoxy-tryptoline in rat pineal gland: Light-dark related studies

High resolution selected ion monitoring of 5-methoxy-tryptoline in rat pineal gland: Light-dark related studies

Determination of 17-hydroxyprogesterone in plasma by gas chromatography—mass spectrometry with high-resolution selected-ion monitoring

A method for determining 17-hydroxyprogesterone in plasma by isotope dilution-mass spectrometry is described. For the internal standard 17-hydroxy[ 2H 4]progesterone is used. Extraction of plasma is followed by conversion into the 3,20-dienol,17-tristrimethylsilyl ether derivative and analysis by capillary gas chromatography—mass spectrometry with selected-ion monitoring, at a resolution of 6000. The lower limit of quantitation was 1 pg, judged from a criterion of a signal-to-noise ratio of 10. The precision and accuracy of the method were satisfactory.

Diethylhydrogensilyl-cyclic diethylsilylene derivatives in gas chromatography-mass spectrometry of prostanoids : III. F α-prostaglandins and thromboxane B 2 derivatives

The gas chromatographic (GC) and GC-mass spectrometric properties of the diethylhydrogensilyl-cyclic diethylsilylene (DEHS-DES) derivatives of prostaglandin (PG) F 1α methyl ester, PGF 2α methyl ester, 6-keto-PGF 1α methyl ester-alkyloxime and thromboxane (TX) B 2 methyl ester-alkyloxime and the DES derivative of 13,14-dihydro-15-keto-PGF 2α methyl ester-alkyloxime were studied. When the ketonic PGs and TXB 2 were converted into their methyloxime derivatives, the methylene unit values of these five prostanoid derivatives were slightly greater than those of the corresponding dimethylethylsilyl ether derivatives. When the ketonic PGs were converted into their corresponding ethyloxime derivatives, baseline separation was achieved in 20 min by use of a methylsilicone cross-linked fuse-silica capillary column. The mass spectra of these derivatives were characterized by the ion at m/z 157 for F α prostaglandins and m/z 269 for TXB 2. The major fragmentations were directed by the DES group, and other fragmentations common to the prostanoid derivatives were losses of an ethyl radical at the silicon atom, C 5H 11 hydrocarbon fragment, diethylhydrogensilanol and C 15–C 20 hydrocarbon fragment. The mass fragmentations of these prostanoid derivatives are briefly discussed. GC with high-resolution selected-ion monitoring was carried out for the TXB 2 derivative at a resolution of 8000 by monitoring the ion at m/z 269.1573. A 25-pg amount of this derivative showed a well shaped doublet with a signal-to-noise ratio of more than 300:1.

Gas chromatographic—mass spectrometric determination of ethyl carbamate in alcoholic beverages

A sensitive and specific method based on gas chromatography—mass spectrometry for the quantitative determination of ethyl carbamate in table wines, fortified wines (such as ports and sherries), distilled spirits, brandies and liqueurs has been developed. Three characteristic ions from ethyl carbamate [ m/z 89 (molecular ion), 74 and 62] were monitored in the selected-ion monitoring (SIM) mode. The lowest detection limit (based on the response on the m/z 62 ion) was estimated to be 0.5 ng/g (ppb). Additional confirmation techniques, including high-resolution SIM, and methane or isobutane chemical ionization are described.