Simple method for the quantitative analysis of endogenous folate catabolites p-aminobenzoylglutamate (pABG) and its acetamido (apABG) derivative in human serum and urine by liquid chromatography-tandem mass spectrometry

OBJECTIVE: To develop a routine method for quantitative measurement of the folate catabolites p-aminobenzoylglutamate (pABG) and acetamidobenzoylglutamate (apABG) in serum and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). DESIGN AND METHODS: Urine, serum and aqueous standards were thawed. Two microliters of d3-glutamic acid (d3-Glu; 1 mmol/L) was added to 200 uL of specimen as internal standard. The samples were acidified with 4 uL 6N HCL, and aliquots were precipitated with 2 volumes (412 uL) of acetonitrile. For urine specimens 30 volumes (6.18 mL) of acetonitrile was used. Samples were centrifuged at 1900 x g for 10 min and the supernatant (10 microL) injected into a Biorad CAT/MET analytical column fitted to the LC-MS/MS. Detection of the catabolites was by selective multiple ion monitoring (multiple SRM) of the respective transitions. Urine and serum samples were analysed in a group of healthy volunteers and in anonymous samples from patients being tested for PTH and urinary catecholamines. RESULTS: pABG and apABG eluted at 5.2 and 4.74 min, respectively while the d3-glutamic acid eluted at around 7 min. Limit of quantitation (LOQ) for both catabolites was 10 nmol/L (which is equivalent to 33.3 fmol for a 10 microL injection). Limit of detection (LOD) was 1 nmol/L based on a signal to noise ratio of 5:1. A linear calibration curve was obtained from 10 to 100 nmol/L for serum specimens and from 10 to 200 micromol/L for urines. Imprecision for spiked serum samples (n=10) was between 2.5 and 20% for apABG and 4.5 and 21% for pABG (at 10 and 100 nmol/L, respectively). Imprecision for spiked urine samples (n=10) was between 2.9 and 4.0% for apABG and 6.0-12.7% for pABG. Recoveries were between 80 and 122% for serum samples and between 92 and 102% for urine specimens. Total folate catabolites in random urine samples from volunteers (n=5) are 2.9+/-2.3 umol/L (mean+/-S.D.). This group also had total serum catabolites of 11.9+/-7.6 nmol/L and serum folate of 35.3+/-5.8 nmol/L. Serum from patients being tested for PTH (n=11) had serum folate levels of 27.0+/-10.4 nmol/L with total serum catabolites of 20.4+/-23.8 nmol/L. Levels of serum folate and total catabolites in pregnant women (n=18) were 33.9+/-22.7 and 11.4+/-8.7 nmol/L, respectively. Mean urinary folate catabolites in patients being tested for urinary catecholamines (n=19) was 581.8+/-368.4 nmol/L. CONCLUSION: A simple, reliable and highly specific method by LC-MS/MS for detecting and quantifying the folate catabolites pABG and apABG was developed. This enables, for the first time, the routine clinical analysis of folate utilization in patients.     

A novel alignment method and multiple filters for exclusion of unqualified peptides to enhance label-free quantification using peptide intensity in LC-MS/MS

Though many software packages have been developed to perform label-free quantification of proteins in complex biological samples using peptide intensities generated by LC-MS/MS, two critical issues are generally ignored in this field: (i) peptides have multiple elution patterns across runs in an experiment, and (ii) many peptides cannot be used for protein quantification. To address these two key issues, we have developed a novel alignment method to enable accurate peptide peak retention time determination and multiple filters to eliminate unqualified peptides for protein quantification. Repeatability and linearity have been tested using six very different samples, i.e., standard peptides, kidney tissue lysates, HT29-MTX cell lysates, depleted human serum, human serum albumin-bound proteins, and standard proteins spiked in kidney tissue lysates. At least 90.8% of the proteins (up to 1,390) had CVs ≤ 30% across 10 technical replicates, and at least 93.6% (up to 2,013) had R(2) ≥ 0.9500 across 7 concentrations. Identical amounts of standard protein spiked in complex biological samples achieved a CV of 8.6% across eight injections of two groups. Further assessment was made by comparing mass spectrometric results to immunodetection, and consistent results were obtained. The new approach has novel and specific features enabling accurate label-free quantification.     

Selective and sensitive determination of amisulpride in human plasma by liquid chromatography-tandem mass spectrometry with positive electrospray ionisation and multiple reaction monitoring

The development of a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with positive electrospray ionisation (ESI(+)) and multiple reaction monitoring (MRM) for the selective and sensitive bioanalytical determination of amisulpride, a substituted benzamide derivative, in human plasma is described. Plasma was cleaned up using a liquid-liquid extraction (diisopropylether:dichloromethane=1:1 (v/v)) procedure. The chemically related drug sulpiride was used as internal standard (ISTD) and a primary calibration function was established in the concentration range of 0.50-500.52 ng/ml for amisulpride in plasma by triple analysis of the corresponding calibration standards. A linear relationship between concentration and signal intensity (given as peak area ratio analyte/ISTD) was obtained (linear regression: r=0.9999). A lower limit of quantification (LLQ) of 0.50 ng/ml was used during measurement of study plasma samples. Satisfying results of within-day precision (CV=0.79 to 1.98%) and accuracy (mean relative deviation: -1.68 to 3.58%) and between-day precision (CV=1.34 to 4.62%) and accuracy (mean relative deviation: -1.73 to -3.77%) as well as of recovery (amisulpride: 81.74 to 84.83%; sulpiride: 58.65%) and selectivity investigations confirmed the high reliability of this established LC-MS/MS method. Sufficient stability of amisulpride in plasma was achieved during freeze-thaw-cycles, for storage periods of 24h at room temperature and 20 days at <-20 degrees C as well as in extracts (storage conditions: <-20 degrees C, 6 days and 7 degrees C, 6 days) with mean relative deviations between - 2.83 and 2.91%. An example of a pharmacokinetic profile determined after administration of an amisulpride 200mg dose in a pilot study is given in this paper. A peak plasma concentration (C(max)) of 522.58 ng/ml was achieved after 3.55 h (t(max)). Corresponding values of areas under the plasma concentration-time curve (AUC) of 3405.35 ngh/ml (AUC(0-infinity)) and 3306.54 ngh/ml (AUC(0-tlast)) were obtained. The terminal plasma elimination half-life (t(1/2)) was 10.36 h.     

Automatic method for quantitation of mercury in blood, plasma and urine.

Here we report our experience of quantification of mercury in blood, plasma and urine by using modifications of a procedure for cold vapour atomic absorption. We have tried: (1) modifications of the instrumentation including the tower, the cell and apparatus for measurement; (2) to increase the volume of sample, avoiding problems caused by foaming and background to arrive at a reliable method with low detection limit. Blood and plasma samples were digested overnight in a mixture of nitric acid and perchloric acid (1:5). Recovery of known additions of mercury was close to 100%. Coefficients of variation (CV) within runs and between runs was for B-Hg 4.7 and 9.5, respectively at 20 nmol/l, and for U-Hg 1.8 and 5.2, respectively at 57 nmol/l. The same detection limit of 5 nmol/l was obtained with blood, plasma and urine. This is in the lower range of non-occupationally exposed normal subjects. The results, including those obtained in sample exchange with other laboratories and with reference materials, indicate that the accuracy of this method for quantification of mercury is good.     

High-throughput and simultaneous measurement of homocysteine and cysteine in human plasma and urine by liquid chromatography-electrospray tandem mass spectrometry

Total homocysteine (tHcy) and cysteine (tCys) concentrations in biological fluids are routinely used in the clinical diagnosis of genetic and metabolic diseases, and this necessitates the development of rapid and sensitive methods for quantification. Liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) was used to measure tHcy and tCys in 23 plasma and 21 urine samples from healthy adults and 14 urine samples from healthy children. The results were compared with a standard high-performance liquid chromatography (HPLC) method. The coefficient of variation (CV) for the LC-MS/MS method ranged from 2.9% to 6.1% for the intraassay and 4.8% to 6.4% for the interassay. Mean recoveries were close to 100% for both plasma and urinary tHcy and tCys. The mean plasma tHcy and tCys concentrations in healthy adults were 8.62 and 261.40 micromol/L, respectively. The mean urinary tHcy and tCys in adults were 0.98 and 22.60 micromol/mmol creatinine, respectively. The mean urinary tHcy and tCys in children were 1.17 and 27.43 micromol/mmol creatinine, respectively. Bland-Altman difference plots of method comparison between LC-MS/MS and HPLC showed good agreement in plasma and urinary tHcy and tCys concentrations. Our method is suitable for rapid measurements, and the reported urinary values in children will help to develop a pediatric reference range for clinical use.     

Determination of thiopental in urine sample with high-performance liquid chromatography using iodine-azide reaction as a postcolumn detection system

The reaction between iodine and azide ions induced by thiopental was utilized as a postcolumn reaction for chromatographic determination of thiopental. The method is based on the separation of thiopental on an Nova-Pak CN HP column with an acetonitrile-aqueous solution of sodium azide as a mobile phase, followed by spectrophotometric measurement of the residual iodine (lambda=350 nm) from the postcolumn iodine-azide reaction induced by thiopental after mixing an iodine solution containing iodide ions with the column effluent containing azide ions and thiopental. Chromatograms obtained for thiopental showed negative peaks as a result of the decrease in background absorbance. The detection limit (defined as S/N=3) was 20 nM (0.4 pmol injected amount) for thiopental. Calibration graphs, plotted as peak area versus concentrations, were linear from 40 nM. The elaborated method was applied to determine thiopental in urine samples. The detection limit (defined as S/N=3) was 0.025 nmol/ml urine. Calibration graphs, plotted as peak area versus concentrations, were linear from 0.05 nmol/ml urine. Authentic urine samples were analyzed, thiopental was determined at nmol/ml urine level.     

Optimization for peptide sample preparation for urine peptidomics

Analysis of native or endogenous peptides in biofluids can provide valuable insights into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for non-invasive monitoring of human diseases. The non-invasive nature of urine collection and the abundance of peptides in the urine makes analysis by high-throughput ‘peptidomics’ methods , an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regards to difficulties associated with sample preparation. The urine matrix can provide significant background interference in making the analytical measurements that it hampers both the identification of peptides and the depth of the peptidomics read when utilizing LC-MS based peptidome analysis. We report on a novel adaptation of the standard solid phase extraction (SPE) method to a modified SPE (mSPE) approach for improved peptide yield and analysis sensitivity with LC-MS based peptidomics in terms of time, cost, clogging of the LC-MS column, peptide yield, peptide quality, and number of peptides identified by each method. Expense and time requirements were comparable for both SPE and mSPE, but more interfering contaminants from the urine matrix were evident in the SPE preparations (e.g., clogging of the LC-MS columns, yellowish background coloration of prepared samples due to retained urobilin, lower peptide yields) when compared to the mSPE method. When we compared data from technical replicates of 4 runs, the mSPE method provided significantly improved efficiencies for the preparation of samples from urine (e.g., mSPE peptide identification 82% versus 18% with SPE; p = 8.92E-05). Additionally, peptide identifications, when applying the mSPE method, highlighted the biology of differential activation of urine peptidases during acute renal transplant rejection with distinct laddering of specific peptides, which was obscured for most proteins when utilizing the conventional SPE method. In conclusion, the mSPE method was found to be superior to the conventional, standard SPE method for urine peptide sample preparation when applying LC-MS peptidomics analysis due to the optimized sample clean up that provided improved experimental inference from the confidently identified peptides.     

Determination of cadmium in blood, plasma, and urine by electrothermal atomic absorption spectrophotometry after isolation by anion-exchange chromatography

The determination of cadmium in whole blood, urine, or plasma by atomic absorption using electrothermal atomization is described. In preparation for atomic absorption analysis, cadmium was concentrated on an anion-exchange column, significantly lowering the limit of detection and allowing for the first time the accurate and precise determination of plasma cadmium concentrations in persons/animals with low-level cadmium exposures. Recovery of 109Cd from spiked whole blood, plasma, and urine into supernatants of nitric acid-deproteinated samples averaged 99, 100, and 95%, respectively. Anion-exchange isolation of the anionic chlorocadmium complex removed 99.8% of the major elements associated with a deproteinated whole blood sample. The recovery of 109Cd from the anion-exchange column was 92.2 +/- 0.9% (mean +/- SE, N = 35). The separation of cadmium from constituents in blood, urine, or plasma in this manner allowed comparison of unknown samples to aqueous standards with a defined acid matrix using commercially available acids. The mean intra-assay coefficient of variation (CV) was 12 +/- 3% (mean +/- SE, N = 6) for blood, plasma, and urine samples having cadmium concentrations of 0.1-0.8 microgram/liter. The interassay CV was 13% (N = 7) for a blood sample containing 0.6 microgram Cd/liter. The recovery of known amounts of cadmium added to blood, plasma, and urine in the range of 0.2 to 5.0 micrograms Cd/liter was 97 +/- 6% (mean +/- SE, N = 4).     

Determination of nicotine and cotinine in tobacco harvesters' urine by solid-phase extraction and liquid chromatography

A solid-phase extraction method using Drug Test-1 column containing chemically modified silica as a solid support for sample clean up and reversed phase ion-paired high-pressure liquid chromatography method have been developed for the simultaneous determination of nicotine and its metabolite cotinine from the urine samples. Mobile phase was consisted of acetate buffer (containing 0.03 M sodium acetate and 0.1 M acetic acid) pH 3.1 and acetonitrile (78:22% (v/v)) containing 0.02 M sodium octanosulfonate as an ion pair agent. pH of the mobile phase was adjusted to 3.6 with triethylamine for better resolution and to prevent peak tailing. The linearity was obtained in the range of 0.5-10 microg/ml concentrations of nicotine and cotinine standards. The correlation coefficients were 0.998 for cotinine and 0.999 for nicotine. The recoveries were obtained in the range of 79-97% with average value of 85% for nicotine and in the range of 82-98% with average value of 88% for cotinine. The limit of detection was 2 ng/ml for cotinine and 5 ng/ml for nicotine with 2 ml urine for extraction, calculated by taking signal to noise ratio 10:3. The intra-day co-efficient of variation (CV) were <4 and 7% and inter-day CV were <9 and 7% for nicotine and cotinine, respectively. The method was applied to the urine samples of tobacco harvesters, who suffer from green tobacco sickness (GTS) to check the absorption of nicotine through dermal route during the various processes of tobacco cultivation due to its good reproducibility and sensitivity.     

Precision enhancement of MALDI-TOF MS using high resolution peak detection and label-free alignment

We have developed an automated procedure for aligning peaks in multiple TOF spectra that eliminates common timing errors and small variations in spectrometer output. Our method incorporates high-resolution peak detection, re-binning, and robust linear data fitting in the time domain. This procedure aligns label-free (uncalibrated) peaks to minimize the variation in each peak's location from one spectrum to the next, while maintaining a high number of degrees of freedom. We apply our method to replicate pooled-serum spectra from multiple laboratories and increase peak precision (t/sigma(t)) to values limited only by small random errors (with sigma(t) less than one time count in 89 out of 91 instances, 13 peaks in seven datasets). The resulting high precision allowed for an order of magnitude improvement in peak m/z reproducibility. We show that the CV for m/z is 0.01% (100 ppm) for 12 out of the 13 peaks that were observed in all datasets between 2995 and 9297 Da.     

Simultaneous determination of hydroxycinnamates and catechins in human urine samples by column switching liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry

A quantitative liquid chromatography mass spectrometry (LC-MS) methodology with online sample clean up by column switching is described for the simultaneous determination of the hydroxycinnamates, caffeic acid and chlorogenic acid, and of the catechins, epicatechin and catechin in human urine samples. Enzymatically treated urine samples were directly injected onto the LC-MS system, where sample clean up was performed by a reversed-phase Zorbax 300SB C(3) column and selective elution of the target compounds onto a Zorbax SB C(18) column resulted in final separation prior to detection by atmospheric pressure chemical ionization (APCI) MS using single ion monitoring (SIM) in negative mode. Linear calibration graphs were achieved in the dynamic range of 10-1000 ng/ml urine. The inter- and intraassay coefficients of variation (C.V.%) for the analysis of the four compounds in quality control urine samples were between 7.8 and 10.9, n=17 (reproducibility), and the repeatability of the assay was between 2.5 and 5.0% (n=12). Analyses of urine samples from a human dietary intervention study with intake of 600 g of fruits and vegetables were demonstrated. To our knowledge, this is the first method described that allows simultaneous determination of both hydroxycinnamates and catechins in biological samples.     

Determination of urinary 5-hydroxytryptophol glucuronide by liquid chromatography-mass spectrometry

5-Hydroxytryptophol glucuronide (GTOL) is the major excretion form of 5-hydroxytryptophol (5-HTOL), a minor serotonin metabolite under normal conditions. Because the concentration of 5-HTOL is markedly increased following consumption of alcohol, measurement of 5-HTOL is used as a sensitive biomarker for detection of recent alcohol intake. This study describes the development and evaluation of a liquid chromatography-electrospray ionization mass spectrometry (LC-MS) procedure for direct quantification of GTOL in human urine. Deuterium labelled GTOL (GTOL-(2)H(4)) was used as internal standard. GTOL was isolated from urine by solid-phase extraction on a C(18) cartridge prior to injection onto a gradient eluted Hypurity C(18) reversed-phase HPLC column. The detection limit of the method was 2.0 nmol/L and the measuring range 6-8500 nmol/L. The intra- and inter-assay coefficients of variation were <3.5% (n=10) and <6.0% (n=9), respectively. The new LC-MS method was highly correlated with an established GC-MS method for urinary 5-HTOL (r(2)=0.99, n=70; mean 5-HTOL/GTOL ratio=1.10). This is the first direct assay for quantification of GTOL in urine. The method is suitable for routine application.     

Variability in low-molecular subproteome of blood serum of healthy man in normal life conditions

For analysis of inter-individual variability in low-molecular serum subproteome proteome profiles of healthy men at the age of 20-30 years (36 subjects), 30-40 years (11 subjects) and 40-50 years (11 subjects) were obtained. Serum samples were fractionated on magnetic beads MB WCX using ClinProt robot prior to mass-spectrometry based profiling. Mass-spectra were obtained with time-of-flight mass-spectrometer Autoflex III ("Bruker Daltonics") in automatic mode. It was shown that low-molecular serum subproteome of healthy humans was characterized by significant inter-individual variability. 21% of all peaks in proteome profiles had coefficient of variation more than 50% and 29% of all peaks had low dispersion (CV < 30%).Therefore majority of peaks in proteome profile were peaks with moderate inter-individual variability (CV from 30% to 50%). Fragments of high-molecular kininogen, inter-alpha-trypsin inhibitor, complement components C3 and C4a, apolipoprotein CI, platelet factor IV, beta2-microglobulin and cystatin C showed wide variation among examined groups of healthy men. Dispersion of high-molecular kininogen, inter-alpha-trypsin inhibitor, apolipoproteins AII and CIII peaks increased with age.     

Determination of iothalamate in rat urine,plasma, and tubular fluid by capillary electrophoresis

A method for the quantitative determination of iothalamate (IOT) in rat urine, plasma and tubular fluid by capillary zone electrophoresis (CE) has been developed and validated. Samples of urine and tubular fluids were diluted with water and samples of plasma were deproteinized with two volumes of acetonitrile containing the internal standard, p-aminobenzoic acid (PABA). A BioFocus 2000 system (Bio-Rad, Hercules, CA, USA) was used. The UV detector was set at 254 nm. The samples were loaded into uncoated fused-silica capillary (40 cm×50 μm) by pressure injection. A borate buffer [20 mM, pH 12 (pH adjusted with 1.0 M NaOH)] was used as the electrophoretic buffer. The typical analytical conditions were: voltage, 22 kV; injection, 9 psi×s; capillary and carousel temperatures were 20°C and 18°C respectively. The linear relationship was observed between time-corrected peak area of IOT in water and urine or the corrected peak area ratio of IOT to PABA in plasma and the nominal concentration of IOT with correlation coefficient greater than 0.999. The intra- and inter-day coefficients of variation (CV) were less than 8%. The concentration of IOT in plasma, urine and tubular fluid determined by CE can be used for estimation of whole kidney and single nephron clearances.     

Quantification of acetylcholine, choline, betaine, and dimethylglycine in human plasma and urine using stable-isotope dilution ultra performance liquid chromatography-tandem mass spectrometry

Disorders in choline metabolism are related to disease conditions. We developed a stable-isotope dilution ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of acetylcholine (ACh), betaine, choline, and dimethylglycine (DMG). We used this method to measure concentrations of the analytes in plasma and urine in addition to other biological fluids after a protein precipitation by acetonitrile. The detection limits were between 0.35 nmol/L (for ACh in urine) and 0.34 μmol/L (for betaine in urine). ACh concentrations were not detectable in plasma. Intraassay and interassay coefficient of variation (CVs) were all <10.0% in biological fluids, except for DMG in cerebrospinal fluid (CV=12.44%). Mean recoveries in urine pool samples were between 99.2% and 103.9%. The urinary excretion of betaine, choline, and DMG was low, with approximately 50.0% higher excretion of choline in females compared to males. Median urinary excretion of ACh were 3.44 and 3.92 μmol/mol creatinine in males and females, respectively (p=0.689). Plasma betaine concentrations correlated significantly with urinary excretions of betaine (r=0.495, p=0.027) and choline (r=0.502, p=0.024) in females. Plasma choline concentrations correlated significantly with urinary excretion of ACh in males (r=0.419, p=0.041) and females (r=0.621, p=0.003). The new method for the simultaneous determination of ACh, betaine, choline, and DMG is sensitive, precise, and fast enough to be used in clinical investigations related to the methylation pathway.     

Analysis of urinary proteins from 50 patients with multiple myeloma by discelectrophoresis (author's transl)]

Urinary proteins from 50 patients with multiple myeloma (37 Ig G, 6 Ig A, 7 Bence Jones) were investigated by discelectrophoresis in polyacrylamidgels containing sodium dodecylsulfat. All samples were also characterized by immunelectrophoresis. Quantitatively and qualitatively normal proteinuria was found in 13 patients (26%). 22 patients (44%) had monoclonal free light chains in the urine, kappachains were eliminated mainly in the monomeric form, lambdachains in all samples in the dimeric form. In 2 patients were found to exist light chains as monomers and dimers. 11 other patients (22%) had peaks of monoclonal Ig G or Ig A in the urine, always associated with the elimination of other nonmonoclonal proteins. Non-specific proteinuria was found in the remaining 4 patients. The clinical validity of the findings is discussed.     

Ryanodine receptors, a family of intracellular calcium ion channels, are expressed throughout early vertebrate development

Background

We developed a new version of the open source software package Peptrix that can yet compare large numbers of Orbitrap? LC-MS data. The peptide profiling results for Peptrix on MS1 spectra were compared with those obtained from a small selection of open source and commercial software packages: msInspect, Sieve? and Progenesis?. The properties compared in these packages were speed, total number of detected masses, redundancy of masses, reproducibility in numbers and CV of intensity, overlap of masses, and differences in peptide peak intensities. Reproducibility measurements were taken for the different MS1 software applications by measuring in triplicate a complex peptide mixture of immunoglobulin on the Orbitrap? mass spectrometer. Values of peptide masses detected from the high intensity peaks of the MS1 spectra by peptide profiling were verified with values of the MS2 fragmented and sequenced masses that resulted in protein identifications with a significant score.

Findings

Peptrix finds about the same number of peptide features as the other packages, but peptide masses are in some cases approximately 5 to 10 times less redundant present in the peptide profile matrix. The Peptrix profile matrix displays the largest overlap when comparing the number of masses in a pair between two software applications. The overlap of peptide masses between software packages of low intensity peaks in the spectra is remarkably low with about 50% of the detected masses in the individual packages. Peptrix does not differ from the other packages in detecting 96% of the masses that relate to highly abundant sequenced proteins. MS1 peak intensities vary between the applications in a non linear way as they are not processed using the same method.

Conclusions

Peptrix is capable of peptide profiling using Orbitrap? files and finding differential expressed peptides in body fluid and tissue samples. The number of peptide masses detected in Orbitrap? files can be increased by using more MS1 peptide profiling applications, including Peptrix, since it appears from the comparison of Peptrix with the other applications that all software packages have likely a high false negative rate of low intensity peptide peaks (missing peptides).     

pParse: a method for accurate determination of monoisotopic peaks in high-resolution mass spectra

Determining the monoisotopic peak of a precursor is a first step in interpreting mass spectra, which is basic but non-trivial. The reason is that in the isolation window of a precursor, other peaks interfere with the determination of the monoisotopic peak, leading to wrong mass-to-charge ratio or charge state. Here we propose a method, named pParse, to export the most probable monoisotopic peaks for precursors, including co-eluted precursors. We use the relationship between the position of the highest peak and the mass of the first peak to detect candidate clusters. Then, we extract three features to sort the candidate clusters: (i) the sum of the intensity, (ii) the similarity of the experimental and the theoretical isotopic distribution, and (iii) the similarity of elution profiles. We showed that the recall of pParse, MaxQuant, and BioWorks was 98-98.8%, 0.5-17%, and 1.8-36.5% at the same precision, respectively. About 50% of tandem mass spectra are triggered by multiple precursors which are difficult to identify. Then we design a new scoring function to identify the co-eluted precursors. About 26% of all identified peptides were exclusively from co-eluted peptides. Therefore, accurately determining monoisotopic peaks, including co-eluted precursors, can greatly increase peptide identification rate.     

F(2)-isoprostane and prostaglandin F(2 alpha)metabolite excretion rate and day to day variation in healthy humans.

Isoprostanes are mainly formed in vivo by a non-enzymatic free radical catalysed oxidation of arachidonic acid. Studies have indicated that a major isoprostane, 8-iso-PGF(2 alpha)in plasma and urine is a reliable biomarker of oxidative stress. Prostaglandins are formed by enzymatic oxidation of arachidonic acid catalysed by cyclooxygenase (COX). 15-Keto-dihydro-PGF(2 alpha), a major metabolite of prostaglandin F(2 alpha)in plasma, and also found in urine, is considered to be a useful biomarker of inflammation. To investigate the excretion pattern and day to day variation of 8-iso-PGF(2 alpha)and 15-keto-dihydro-PGF(2 alpha)in healthy individuals, morning urine samples were collected from 13 volunteers on 10 successive days. The samples were analysed for free 8-iso-PGF(2 alpha)and 15-keto-dihydro-PGF(2 alpha)by radioimmunoassay. The mean excretion rate of 8-iso-PGF(2 alpha)was 0.27+/-0.11 nmol/mmol creatinine (mean+/-SD, n=13) and the coefficient of variation was 42% during the 10 days. The mean excretion rate of 15-keto-dihydro-PGF(2 alpha)was 0.46+/-0.19 nmol/mmol creatinine, giving a coefficient of variation of 41%. The mean values of 8-iso-PGF(2 alpha)were significantly correlated with the mean values of 15-keto-dihydro-PGF(2 alpha)(r=0.68, P=0.01). In conclusion, day to day biological variation in urinary excretion rate of 8-iso-PGF(2 alpha)and 15-keto-dihydro-PGF(2 alpha)should be taken into account in evaluating a clinical study unless a large increase or decrease of these parameters has been obtained.     

Excretion of corticosteroid metabolites in urine and faeces of rats.

Stress enhances the production of corticosteroids by the adrenal cortex, resulting in the increased excretion of their metabolites in urine and faeces. An intraperitoneal injection of radioactive corticosterone was applied to adult, male Sprague-Dawley rats to monitor the route and delay of excreted metabolites in urine and faeces. Peak concentrations appeared in urine after 3.2 +/- 1.9 h and in faeces after 16.7 +/- 4.3 h. Altogether about 20% of the recovered metabolites were found in urine and about 80% in faeces. Using high-performance liquid chromatography (HPLC), several peaks of radioactive metabolites were found. Some metabolites were detected by enzyme immunoassay (EIA) using two different antibodies (corticosterone, 11beta-OH-aetiocholanolone). There was a marked diurnal variation with low levels of faecal corticosterone metabolites in the evening and higher values in the morning. This diurnal variation was influenced neither by the intraperitoneal injection of isotonic saline nor by ACTH. However, the administration of dexamethasone eliminated the morning peak for 2 days.