Stable Isotope Dilution Method Research Articles

Fate of Fusarium Toxins during the Malting Process.

Little is known about the fate of Fusarium mycotoxins during the barley malting process. To determine the fungal DNA and mycotoxin concentrations during malting, we used barley grain harvested from field plots that we had inoculated with Fusarium species that produce type A or type B trichothecenes or enniatins. Using a recently developed multimycotoxin liquid chromatography-tandem mass stable isotope dilution method, we identified Fusarium-species-specific behaviors of mycotoxins in grain and malt extracts and compared toxin concentrations to amounts of fungal DNA in the same samples. In particular, the type B trichothecenes and Fusarium culmorum DNA contents were increased dramatically up to 5400% after kilning. By contrast, the concentrations of type A trichothecenes and Fusarium sporotrichioides DNA decreased during the malting process. These data suggest that specific Fusarium species that contaminate the raw grain material might have different impacts on malt quality.

Quantitation of 5-Methyltetrahydrofolate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.

We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF) as a clinical diagnostic test. 5-MTHF is the main biologically active form of folic acid and is involved in regulation of homocysteine and DNA synthesis. Measurement of 5-MTHF in CSF provides diagnostic information regarding diseases affecting folate metabolism within the central nervous system, in particular inborn errors of folate metabolism. Determination of 5-MTHF in CSF (50 μL) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). 5-MTHF in CSF is determined by a 1:2 dilution with internal standard (5-MTHF-(13)C5) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve (25-400 nM) and has an analytical measurement range of 3-1000 nM.

Quantitation of S-Adenosylmethionine and S-Adenosylhomocysteine in Plasma Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.

We describe a simple stable isotope dilution method for accurate determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma as a diagnostic test. SAM and SAH are key metabolic intermediates of methionine metabolism and the methylation cycle. Determination of SAM and SAH in plasma was performed by high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Calibrators (SAM and SAH) and internal standards ((2)H3-SAM and (2)H4-SAH) were included in each analytical run for calibration. Sample preparation involved combining 20 μL sample with 180 μL of internal standard solution consisting of heavy isotope labeled internal standards in mobile phase A and filtering by ultracentrifugation through a 10 kd MW cutoff membrane. Sample filtrate (3 μL) was injected by a Shimadzu Nexera LC System interfaced with a 5500 QTRAP(®) (AB Sciex). Chromatographic separation was achieved on a 250 mm × 2.0 mm EA:faast column from Phenomenex. Samples were eluted at a flow rate of 0.20 mL/min with a binary gradient with a total run time of 10 min. The source operated in positive ion mode at an ion spray voltage of +5000 V. SAM and SAH resolved by a gradient to 100 % methanol with retention times of 6.0 and 5.7 min, respectively. The observed m/z values of the fragment ions were m/z 399 → 250 for SAM, m/z 385 → 136 for SAH, m/z 402 → 250 for (2)H3-SAM, m/z 203 → 46. The calibration curve was linear over the ranges of 12.5-5000 nmol/L for SAM and SAH.

Quantification of γ-Aminobutyric Acid in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.

We describe a simple stable isotope dilution method for accurate and precise measurement of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter in human cerebrospinal fluid (CSF) as a clinical diagnostic test. Determination of GABA in CSF (50 μL) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analysis of free and total GABA requires two individual sample preparations and mass spectrometry analyses. Free GABA in CSF is determined by a 1:2 dilution with internal standard (GABA-D2) and injected directly onto the HPLC-ESI-MS/MS system. Determination of total GABA in CSF requires additional sample preparation in order to hydrolyze all the bound GABA in the sample to the free form. This requires hydrolyzing the sample by boiling in acidic conditions (hydrochloric acid) for 4 h. The sample is then further diluted 1:10 with a 90 % acetonitrile/0.1 % formic acid solution and injected into the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve and is linear from 6 nM to 1000 nM and 0.63 μM to 80 μM for free and total GABA, respectively.

EU marker polycyclic aromatic hydrocarbons in food supplements: analytical approach and occurrence

Several food supplements comprising botanical, oil and bee products collected from retail markets in different countries were tested for the occurrence of 4 EU marker Polycyclic aromatic hydrocarbons (PAHs; benz[a]anthracene, chrysene, benzo[b]fluoranthene and benzo[a]pyrene). A robust GC/MS-based stable-isotope dilution method was used taking into account the differences in the type of matrices. The accuracy of the results was assessed by implementing several quality control tools. Sixty-eight samples of 94 analysed products exceeded the level of 0.5 μg/kg for the sum of the four EU marker PAHs (ΣPAH4). Benzo[a]pyrene exceeded the limit of quantification in 49 samples. The PAH with the highest abundance in all products was chrysene. On average, propolis extracts and other bee products showed relatively high levels of ΣPAH4 (mean 188.2 μg/kg), whereas the contamination levels of fish oil supplements were very low or mostly undetectable. Considerably high ΣPAH4 amounts found in some samples could remarkably increase the daily exposure of consumers to PAHs, demonstrating the need for continuous monitoring of ΣPAH4 in food supplements.

A facile stable-isotope dilution method for determination of sphingosine phosphate lyase activity

A new technique for quantifying sphingosine phosphate lyase activity in biological samples is described. In this procedure, 2-hydrazinoquinoline is used to convert (2E)-hexadecenal into the corresponding hydrazone derivative to improve ionization efficiency and selectivity of detection. Combined utilization of liquid chromatographic separation and multiple reaction monitoring-mass spectrometry allows for simultaneous quantification of the substrate S1P and product (2E)-hexadecenal. Incorporation of (2E)- d5-hexadecenal as an internal standard improves detection accuracy and precision. A simple one-step derivatization procedure eliminates the need for further extractions. Limits of quantification for (2E)-hexadecenal and sphingosine-1-phosphate are 100 and 50fmol, respectively. The assay displays a wide dynamic detection range useful for detection of low basal sphingosine phosphate lyase activity in wild type cells, SPL-overexpressing cell lines, and wild type mouse tissues. Compared to current methods, the capacity for simultaneous detection of sphingosine-1-phosphate and (2E)-hexadecenal greatly improves the accuracy of results and shows excellent sensitivity and specificity for sphingosine phosphate lyase activity detection.

Simultaneous Quantification of Methylated Cytidine and Adenosine in Cellular and Tissue RNA by Nano-Flow Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.

The rising interest in understanding the functions, regulation, and maintenance of the epitranscriptome calls for robust and accurate analytical methods for the identification and quantification of post-transcriptionally modified nucleosides in RNA. Monomethylations of cytidine and adenosine are common post-transcriptional modifications in RNA. Herein, we developed an LC-MS/MS/MS coupled with the stable isotope-dilution method for the sensitive and accurate quantifications of 5-methylcytidine (m(5)C), 2'-O-methylcytidine (Cm), N(6)-methyladenosine (m(6)A), and 2'-O-methyladenosine (Am) in RNA isolated from mammalian cells and tissues. Our results showed that the distributions of m(5)C, Cm and Am are tissue-specific. In addition, the 2'-O-methylated ribonucleosides (Cm and Am) are present at higher levels than the corresponding methylated nucleobase products (m(5)C and m(6)A) in total RNA isolated from mouse brain, pancreas, and spleen but not mouse heart. We also found that the levels of m(5)C, Cm, and Am are significantly lower (by 6.5-43-fold) in mRNA than in total RNA isolated from HEK293T cells, whereas the level of m(6)A was slightly higher (by 1.6-fold) in mRNA than in total RNA. The availability of this analytical method, in combination with genetic manipulation, may facilitate the future discovery of proteins involved in the maintenance and regulation of these RNA modifications.

Effects of resistance training on testosterone metabolism in younger and older men

This study investigated the effects of resistance training (RT) on the metabolism of testosterone (T) in younger (n=5, 28±3yrs.) and older (n=8, 70±2yrs.) men. Experimental heavy resistance exercises (5×10RM leg presses) were performed before and after a 12-month of RT. No age differences were found in the production or metabolic clearance rate of T (determined by stable isotope dilution method), skeletal muscle androgen receptor content or serum LH concentrations due to acute or chronic RT. The T production capacity response to gonadotropin stimulation and the concentrations of the urinary T metabolites (androsterone and etiocholanolone) were lower in the older compared to younger men (p<0.05–0.01). This study further showed that RT may have acute effect on T production and clearance rates, while the exercise-induced increases in serum T appeared to be induced by decreased metabolic clearance rate of T. Attenuated T production capacity and urinary excretion of T metabolites in older men may reflect the known reduction in testicular steroidogenesis upon aging. No changes were observed in T metabolism due to RT indicating a homeostatic stability for this hormone in men of different ages.

Reversible increase in maximal cortisol secretion rate in septic shock.

Cortisol clearance is reduced in sepsis and may contribute to the development of impaired adrenocortical function that is thought to contribute to the pathophysiology of critical illness-related corticosteroid insufficiency. We sought to assess adrenocortical function using computer-assisted numerical modeling methodology to characterize and compare maximal cortisol secretion rate and free cortisol half-life in septic shock, sepsis, and healthy control subjects. Post hoc analysis of previously published total cortisol, free cortisol, corticosteroid-binding globulin, and albumin concentration data. Single academic medical center. Subjects included septic shock (n = 45), sepsis (n = 25), and healthy controls (n = 10). I.v. cosyntropin (250 μg). Solutions for maximal cortisol secretion rate and free cortisol half-life were obtained by least squares solution of simultaneous, nonlinear differential equations that account for free cortisol appearance and elimination as well as reversible binding to corticosteroid-binding globulin and albumin. Maximal cortisol secretion rate was significantly greater in septic shock (0.83 nM/s [0.44, 1.58 nM/s] reported as median [lower quartile, upper quartile]) compared with sepsis (0.51 nM/s [0.36, 0.62 nM/s]; p = 0.007) and controls (0.49 nM/s [0.42, 0.62 nM/s]; p = 0.04). The variance of maximal cortisol secretion rate in septic shock was also greater than that of sepsis or control groups (F test, p < 0.001). Free cortisol half-life was significantly increased in septic shock (4.6 min [2.2, 6.3 min]) and sepsis (3.0 min [2.3, 4.8 min] when compared with controls (2.0 min [1.2, 2.6 min]) (both p < 0.004). Results obtained by numerical modeling are consistent with comparable measures obtained by the gold standard stable isotope dilution method. Septic shock is associated with generally not only higher levels but also greater variance of maximal cortisol secretion rate when compared with control and sepsis groups. Additional studies would be needed to determine whether assessment of cortisol kinetic parameters such as maximal cortisol secretion rate and free cortisol half-life is useful in the diagnosis or management of critical illness-related corticosteroid insufficiency.

Simultaneous measurement of tabun, sarin, soman, cyclosarin, VR, VX, and VM adducts to tyrosine in blood products by isotope dilution UHPLC-MS/MS.

This work describes a new specific, sensitive, and rapid stable isotope dilution method for the simultaneous detection of the organophosphorus nerve agents (OPNAs) tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VR, VX, and VM adducts to tyrosine (Tyr). Serum, plasma, and lysed whole blood samples (50 μL) were prepared by protein precipitation followed by digestion with Pronase. Specific Tyr adducts were isolated from the digest by a single solid phase extraction (SPE) step, and the analytes were separated by reversed-phase ultra high performance liquid chromatography (UHPLC) gradient elution in less than 2 min. Detection was performed on a triple quadrupole tandem mass spectrometer using time-triggered selected reaction monitoring (SRM) in positive electrospray ionization (ESI) mode. The calibration range was characterized from 0.100-50.0 ng/mL for GB- and VR-Tyr and 0.250-50.0 ng/mL for GA-, GD-, GF-, and VX/VM-Tyr (R(2) ≥ 0.995). Inter- and intra-assay precision had coefficients of variation of ≤17 and ≤10%, respectively, and the measured concentration accuracies of spiked samples were within 15% of the targeted value for multiple spiking levels. The limit of detection was calculated to be 0.097, 0.027, 0.018, 0.074, 0.023, and 0.083 ng/mL for GA-, GB-, GD-, GF-, VR-, and VX/VM-Tyr, respectively. A convenience set of 96 serum samples with no known nerve agent exposure was screened and revealed no baseline values or potential interferences. This method provides a simple and highly specific diagnostic tool that may extend the time postevent that a confirmation of nerve agent exposure can be made with confidence.

Elevated levels of pro-inflammatory oxylipins in older subjects are normalized by flaxseed consumption

Background and aimsOxylipins, including eicosanoids, are highly bioactive molecules endogenously produced from polyunsaturated fatty acids. Oxylipins play a key role in chronic disease progression. It is possible, but unknown, if oxylipin concentrations change with the consumption of functional foods or differ with subject age. MethodsTherefore, in a parallel comparator trial, 20 healthy individuals were recruited into a younger (19–28years) or older (45–64years) age group (n=10/group). Participants ingested one muffin/day containing 30g of milled flaxseed (6g alpha-linolenic acid) for 4weeks. Plasma oxylipins were isolated through solid phase extraction, analyzed with HPLC-MS/MS targeted lipidomics, and quantified with the stable isotope dilution method. ResultsAt baseline, the older group exhibited 13 oxylipins ≥2-fold the concentration of the younger group. Specifically, pro-inflammatory oxylipins 5-hydroxyeicosatetraenoic acid, 9,10,13-trihydroxyoctadecenoic acid, and 9,12,13-trihydroxyoctadecenoic acid were significantly greater in the older (1.1±0.23nM, 5.6±0.84nM, and 4.5±0.58nM, respectively) versus the younger group (0.34±0.12nM, 3.5±0.33nM, and 3.0±0.24nM, respectively) (p<0.05). After 4weeks of flaxseed consumption the number of oxylipins that were ≥2-fold higher in the older versus the younger group was reduced to 3. 5-Hydroxyeicosatetraenoic acid, 9,10,13-trihydroxyoctadecenoic acid, and 9,12,13-trihydroxyoctadecenoic acid decreased in the older group to concentrations equivalent to the younger group after flaxseed consumption. ConclusionThese data suggest a potential role for oxylipins in the aging process and how nutritional interventions like flaxseed can beneficially disrupt these biological changes associated with inflammation and aging.

Absolute Quantification of Cytochrome P450 and Uridine-Diphosphate Glucuronosyl Transferase Isoforms by Proteomics-based Approach

Abstract A strategy for the absolute quantitation of metabolic enzymes in rat liver microsomes was developed based on shotgun-based proteomics approach. Rat liver microsomes were digested with trypsin, and drug metabolizing enzymes were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) using standard curves based on custom synthesized specific peptides for each enzyme. The assays for cytochrome P450 (CYP450) and uridine diphosphoglucuronosyl transferase (UGT) showed good linearity ( r > 0.995) with lower limits of quantitation of 10 nM. A synthetic isotope labeled specific peptide was then used as internal standard to determine UGT1A1 by stable isotope dilution method. The labeled peptide behaved similarly to the unlabeled peptide in LC-MS/MS and the assay was linear in matrix solution. The UGT1A1 concentration was detected by the labeled peptide method to be 18.2 pmol mg −1 protein compared with 17.3 pmol mg −1 protein obtained by the standard curve method. Although the consistent results were achieved by the two methods, the stable isotope dilution method was more convenient and more suitable for high throughput determinations in complex systems.

Effects of rapid weight loss and regain on body composition and energy expenditure

Weight-classified athletes need an energy intake plan to accomplish target weight reduction. They have to consider body composition and energy metabolism during rapid weight loss followed by rapid weight regain to achieve their energy intake plan. We investigated the effects of rapid weight loss, followed by weight regain, on body composition and energy expenditure. Ten weight-classified athletes were instructed to reduce their body weight by 5% in 7 days. Following the weight loss, they were asked to try to regain all of their lost weight with an ad libitum diet for 12 h. Food intake was recorded during the baseline, weight loss, and regain periods. Fat mass, total body water, and fat-free dry solids were estimated by underwater weighing and stable isotope dilution methods. A three-component model was calculated using Siri's equation. Basal and sleeping metabolic rates were measured by indirect calorimetry. Body composition and energy expenditure were measured before and after weight loss and after weight regain. Body weight, total body water, and fat-free dry solids were decreased after the weight loss period but recovered after weight regain (p < 0.05). Basal metabolic rate did not change throughout the study. Sleeping metabolic rate decreased significantly during weight loss but recovered after weight regain. Changes in total body water greatly affect body weight during rapid weight loss and regain. In addition, rapid weight loss and regain did not greatly affect the basal metabolic rate in weight-classified athletes.

Distinct eicosanoid profile in exhaled breath condensates from granulomatosis with polyangiitis (Wegener’s) patients

IntroductionGranulomatosis with polyangiitis (GPA, Wegener’s) is asystemic vasculitis affecting mostly small vessels, typicallyinvolving the upper respiratory tract, lungs, and kidneys.Together with microscopic polyangiitis, eosinophilic gran-ulomatosis with polyangiitis (Churg–Strauss syndrome),and renal-limited vasculitis, it commonly shows circulatingantineutrophil cytoplasmic antibodies (ANCA). As such, itis typically referred to as an ANCA-associated vasculitis [1,2]. The pathogenesis of GPA has not been fully elucidated,although the interaction between ANCA and neutrophilsseems pivotal in initiating and supporting an inflammatoryreaction with subsequent cytokine, complement, and endo-thelium involvement [3]. The hallmark of the respiratorytract pathology is granulomatous inflammation, in which theeicosanoid derivatives play an important role. Clinical diag-nostics often requires an invasive approach, i.e., open lungbiopsy, bronchoalveolar lavage, or bronchoscopy-guidedbiopsies. Recently, a noninvasive diagnostic method of therespiratory tract examination was introduced; namely, ex-haled breath condensate (EBC) analysis. We have previous-ly described a highly specific increase in 12-lipoxygenaseproduct (12-HETE) in EBC samples of eosinophilic withpolyangiitis patients [4]. We wondered if ascertainment ofeicosanoid profile in EBC from GPA patients could help innoninvasive diagnostics of GPA.Patients and methodsPatientsWe enrolled 27 patients with GPA treated in the Departmentof Medicine of the University Hospital, JagiellonianUniversity Medical College. The control group consistedof 30 healthy volunteers recruited from the hospital employ-ees with similar gender distribution. GPA was diagnosedaccording to the American college of rheumatology criteria,andall patients werec-ANCA positive [5].The Birminghamvasculitis activity score (BVAS v.3, range 0–63 points) [6]was used to assess activity of GPA; results above threeindicated active disease. All subjects studied gave informedwritten consent to participate. The study protocol compliedwith the Helsinki Declaration and was approved by theJagiellonian University ethics committee.Exhaled breath condensate collection and analysisEBC were collected with EcoScreen I Jaeger® condensingchamber (GmbH Hoechberg, Germany) according to theATS/ERSrecommendations[ 7].CollectedEBC(1–2mlwith-in 15 min) was immediately stored at −80 °C until analysis.High-performance liquid chromatography tandem massspectrometry (HPLC–MS/MS; Qtrap 4000, AppliedBiosystems, Foster City, CA, USA) or gas chromatogra-phy–mass spectrometry (GC–MS; Engine 5989B series IIHewlett Packard, Palo Alto, CA, USA) were used for thequantification of substances with a stable isotope dilutionmethod [8]. The detection levels for the eicosanoids (inpicograms per milliliter) were as previously described [8,9], and palmitic acid (PA)—abundant in EBC—was usedfor a correction of dilution factor. Eicosanoids were

Mild fasting hyperglycemia shifts fuel reliance toward fat during exercise in adults with impaired glucose tolerance

Impaired glucose tolerance (IGT) is characterized by decreased oxidative capacity and reduced carbohydrate utilization during exercise. However, it is unclear if the presence of impaired fasting glucose (IFG) affects fuel utilization during exercise in adults with IGT. We tested the hypothesis that the presence of IFG in adults with IGT decreases reliance on carbohydrate during exercise. Middle-aged, obese, sedentary individuals (n = 6, IGT and n = 6, IFG+IGT) were compared during exercise at 60% peak O2 consumption for 45 min on a cycle ergometer. Glucose rates of appearance and disposal and muscle glycogen were assessed by stable isotope dilution methods, and fat utilization was estimated via indirect calorimetry. A 75-g oral glucose tolerance test was used to determine fasting and 2-h glucose concentrations. A glucose intolerance severity z-score was calculated from the oral glucose tolerance test. Glucose flux (i.e., rates of appearance and disposal) was not different between groups. However, individuals with IFG+IGT had lower muscle glycogen use (P < 0.05) and elevated fat oxidation (P < 0.01) during exercise compared with those with isolated IGT. Plasma nonesterified fatty acids and glucose were significantly higher during exercise in subjects with IFG+IGT vs. IGT alone (P < 0.05). Fat utilization during exercise correlated with fasting glucose (r = 0.57, P = 0.05), glucose intolerance severity z-score (r = 0.66, P = 0.01), and nonesterified fatty acids (trend; r = 0.55, P = 0.08). The presence of IFG shifts fuel selection toward increased fat oxidation and decreased muscle glycogen utilization during exercise in adults with IGT. Whether these differences in substrate use contribute to, or are the result of, movement along the continuum from prediabetes to type 2 diabetes awaits further work.

A stable isotope dilution method for measuring bioavailability of organic contaminants

Methods for determining bioavailability of organic contaminants suffer various operational limitations. We explored the use of stable isotope labeled references in developing an isotope dilution method (IDM) to measure the exchangeable pool (E) of pyrene and bifenthrin as an approximation of their bioavailability in sediments. The exchange of deuterated bifenthrin or pyrene with its native counterpart was completed within 48 h. The derived E was 38–82% for pyrene and 28–59% for bifenthrin. Regression between E and the sum of rapid and slow desorption fractions obtained from sequential desorption showed a slope close to 1.0. The ability of IDM to predict bioavailability was further shown from a strong relationship (r2 > 0.93) between E and bioaccumulation into Chironomus tentans. Given the abundance of stable isotope labeled references and their relatively easy analysis, the IDM has the potential to become a readily adoptable tool for estimating organic contaminants bioaccessibility in various matrices.

A U–HPLC–ESI–MS/MS–Based Stable Isotope Dilution Method for the Detection and Quantitation of Methotrexate in Plasma

High-dose methotrexate (MTX) is used in the treatment of proliferative diseases such as acute lymphoblastic leukemia. Therapeutic drug monitoring of plasma MTX is important to monitor efficacy and adverse events. The authors aimed to develop a liquid chromatography, electrospray ionization, tandem mass spectrometry (LC-ESI-MS/MS)-based method to determine MTX in plasma for therapeutic drug monitoring and pharmacokinetic studies. Samples were analyzed using a Waters Acquity UPLC and Quattro Premier XE. A Waters Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) was used running an isocratic mobile phase of 21% methanol and 10 mM ammonium bicarbonate. The electrospray was operated in the positive ionization mode monitoring the following mass transitions: m/z 455.2 > 308.2 for MTX and m/z 458.2 > 311.2 for MTXd3. The analysis combined straightforward sample preparation, consisting of dilution and protein precipitation, with a 3-minute run time. The method was linear up to 50 μM (r > 0.99), and the coefficient of variation was <6% for intraday and <10% for interday precision. Average recovery was 99%. There were no significant matrix effects. The lower limit of quantitation, defined as the lowest concentration at which the coefficient of variation <20% and S/N > 1:10, was 5 nM. Method comparison with the Abbott TDx fluorescent polarization immunoassay (FPIA) showed excellent agreement, and a small but significant negative constant bias was detected (LC-MS/MS = 0.98 × FPIA - 7.3). [corrected] The authors developed a specific and sensitive stable isotope dilution LC-ESI-MS/MS method to monitor MTX concentrations in plasma within the clinically relevant range. The method can be easily applied in clinical laboratories because it combines straightforward sample pretreatment with LC-MS/MS.

Quantification of a novel sulfhydrylated cGMP in mammalian cells and tissues by liquid chromatography tandem mass spectrometry

8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is formed endogenously under conditions of excess production of NO coupled with oxidative stress. Recently, we found that the electrophilic nitro moiety underwent nucleophilic substitution with hydrogen sulfide anion (HS − ) to yield the novel product 8-SH-cGMP. Endogenous 8-SH-cGMP levels are yet to be identified, however. In this study, we precisely quantified endogenous formation of 8-SH-cGMP in various tissues and cells by means of liquid chromatography- electrospray ionization (ESI)-tandem mass spectrometry (LC–ESI–MS/MS). Mouse tissue homogenates as well as lysates of various mammalian cultured cells were subjected to LC–ESI–MS/MS analysis with a Thermo TSQ vantage triplequadrupole mass spectrometer equipped with a reverse-phase HPLC column. For precise quantification, the stable isotope dilution method was employed: recovery efficiency of the analyte was corrected based on the recovery of the stable isotope-labeled derivative (8-SH-[ 13 C 10 ]cGMP) spiked into the extracts. This method warrants highly sensitive and specific identification of 8-SH-cGMP: detection limit, >1 pmol/ml. According to the LC–MS/MS analyses with stable isotope dilution method, we could quantify the formation of 8-SH-cGMP in mouse tissues and various cells in culture. The highest concentration of 8-SH-cGMP determined in cells was over 10 μM. This sulfhydryl derivative of cGMP is also shown to have a protein kinase G-activating potential with strong resistance to phosphodiesterase-catalyzed degradation. The significant endogenous formation of 8-SH-cGMP therefore may suggest its physiological function as a unique second messenger for hydrogen sulfide.

Label-Free Quantitation of Protein Modifications by Pseudo Selected Reaction Monitoring with Internal Reference Peptides

Liquid chromatography tandem mass spectrometry (LC–MS/MS) based methods provide powerful tools for the quantitative analysis of modified proteins. We have developed a label-free approach using internal reference peptides (IRP) from the target protein for signal normalization without the need for isotope labeling. Ion-trap mass spectrometry and pseudo-selected reaction monitoring (pSRM) were used to acquire full MS/MS and MS3 spectra from target peptides. Skyline, a widely used software for SRM experiments, was used for chromatographic ion extraction. Phosphopeptides spiked into a BSA background yielded concentration response curves with high correlation coefficients (typically >0.9) and low coefficients of variation (≤15%) over a 200-fold concentration range. Stable isotope dilution (SID) and IRP methods were compared for quantitation of six site-specific phosphorylations in the epidermal growth factor receptor (EGFR) in epidermal growth factor-stimulated A431 cells with or without the addition of EGFR inhibitors cetuximab and gefitinib. Equivalent responses were observed with both IRP and SID methods, although analyses using the IRP method typically had higher median CVs (22–31%) than SID (10–20%). Analyses using both methods were consistent with immunoblot using site-selective antibodies. The ease of implementation and the suitability for targeted quantitative comparisons make this method suitable for broad application in protein biochemistry.

Mass spectrometry–based detection and quantification of plasma glycoproteins using selective reaction monitoring

Mass spectrometry-based targeted proteomics is a rapidly expanding method for quantifying proteins in complex clinical samples such as plasma. In conjunction with the stable isotope dilution method, selected reaction monitoring (SRM) assays provide unparalleled sensitivity and selectivity for detection and quantification. A crucial factor for robust SRM assays is the reduction of interference by lowering the background. This can be achieved by the selective isolation of a subproteome, such as N-glycosylated proteins, from the original sample. The present protocol includes the development and optimization of SRM assays associated with each peptide of interest and the qualification of assays in the biological matrix to establish the limits of detection and quantification. The protocol also describes the enrichment of formerly N-glycosylated peptides relying on periodate oxidation of glycan moieties attached to the proteins, their immobilization on solid supports through hydrazide chemistry, proteolysis and enzymatic release of the formerly N-glycosylated peptides.