Mass Isotopomer Abundances Research Articles

Dynamic Proteome Profiling of Protein Fractional and Molar Synthesis Rates in Human Muscle in vivo

This study aimed to develop a method for measuring the abundance and synthesis rate of human muscle proteins in molar rather than fractional (%/d) terms. Losses in proteostasis are implicated in ageing and chronic diseases but research is hindered by a lack of methods for studying protein dynamics in humans. Protein‐specific fractional synthesis rate (FSR) can be measured by stable isotope labelling and peptide mass spectrometry (MS). However, FSR is a measurement of an unknown whole, i.e. protein abundance. If protein abundance changes or is different between conditions, the interpretation of FSR data may be confounded.Three physically active males (21 ± 1 years; height 178 ± 1 cm; weight 75 ± 5 kg) gave their informed consent to the ethically approved procedures. Newly synthesised proteins were labelled by oral consumption of deuterium oxide (50 ml of 99.8 atom %) taken four times per day during days 1–5, and two times per day during days 6–12. Samples of blood and vastus lateralis were collected after an overnight fast on days 0, 4, 8 and 12. Precursor enrichment and incorporation of deuterium oxide into muscle proteins were determined by liquid chromatography tandem MS. Protein abundances were normalised to 30 fmol yeast alcohol dehydrogenase added during sample preparation. FSR (%/d) was calculated from time‐dependent changes in peptide mass isotopomer abundances. FSR and abundance data (fmol/ μg protein) were combined to calculate molar synthesis rates (MSR; fmol/ μg protein/ d).Abundance data were collected for 1,368 proteins that had at least 1 unique peptide. FSR data were calculated from 3,944 peptides representing 940 proteins (average 3 peptides per protein, range 1 – 53 peptides per protein). In total, 769 proteins had abundance and FSR data in two or more participants. The median (M), lower‐(Q1) and upper‐quartile (Q3) values for protein FSR (%/d) were M = 4.39, Q1 = 0.96, Q3 = 7.84. Alpha skeletal muscle actin had the lowest FSR (0.28 ± 0.1 %/d) whereas the highest recorded FSR was 54.43 ± 8.9 %/d (nuclear protein localization protein 4 homolog). tRNA ligases were common amongst top‐ranked proteins by FSR but were ranked amongst the lowest in abundance e.g. Leucine‐‐tRNA ligase was the lowest abundance (5 amol/ μg protein) protein detected. The most abundant protein was myoglobin (1.52 pmol/ μg protein). Average MSR (fmol/ μg protein/ d) was M = 0.05 (Q1 = 0.017, Q3 = 0.13). Metabolic enzymes were amongst the top‐ranked proteins by MSR (range 1–10 fmol/ μg protein/ d) alongside cullin‐associated NEDD8‐dissociated protein 2 (0.9 ± 0.2 fmol/ μg protein/ d) and antioxidant enzymes, including mitochondrial superoxide dismutase [Mn], peroxiredoxin 3, peroxiredoxin 2 and glutathione S‐transferase‐P (each ~0.5 fmol/ μg protein/ d).MSR proteomic data offers new insight to the allocation of cellular resources. Future applications investigating muscle adaptation or differences between health and disease may uncover unexpected insight regarding which proteins are most synthesised and highlight new avenues of research in human muscle physiology.

Reliability of Protein Abundance and Synthesis Measurements in Human Skeletal Muscle

The repeatability of dynamic proteome profiling (DPP), which is a novel technique for measuring the relative abundance (ABD) and fractional synthesis rate (FSR) of proteins in humans, is investigated. LC-MS analysis is performed on muscle samples taken from male participants (n=4) that consumed 4×50mL doses of deuterium oxide (2 H2 O) per day for 14 days. ABD is measured by label-free quantitation and FSR is calculated from time-dependent changes in peptide mass isotopomer abundances. One-hundred one proteins have at least one unique peptide and are used in the assessment of protein ABD. Fifty-four of these proteins meet more stringent criteria and are used in the assessment of FSR data. The median (M), lower-, (Q1 ) and upper-quartile (Q3 ) values for protein FSR (%/d) are M=1.63, Q1 =1.07, and Q3 =3.24, respectively. The technical CV of ABD data has a median value of 3.6% (Q1 1.7% to Q3 6.7%), whereas the median CV of FSR data is 10.1% (Q1 3.5% to Q3 16.5%). These values compare favorably against other assessments of technical repeatability of proteomics data, which often set a CV of 20% as the upper bound of acceptability.

DExSI: a new tool for the rapid quantitation of 13C-labelled metabolites detected by GC-MS.

SummaryStable isotope directed metabolomics is increasingly being used to measure metabolic fluxes in microbial, plant and animal cells. Incorporation of 13C/15N isotopes into a wide range of metabolites is typically determined using gas chromatography-mass spectrometry (GC/MS) or other hyphenated mass spectrometry approaches. The DExSI (Data Extraction for Stable Isotope-labelled metabolites) pipeline is an interactive graphical software package which can be used to rapidly quantitate isotopologues for a wide variety of metabolites detected by GC/MS. DExSI performs automated metabolite annotation, mass and positional isotopomer abundance determination and natural isotope abundance correction. It provides a range of output options and is suitable for high throughput analyses.Availability and implementationDExSI is available for non-commercial use from: https://github.com/DExSI/DExSI/. For Microsoft Windows 7 or higher (64-bit).Supplementary information Supplementary data are available at Bioinformatics online.

Dynamic Proteomics: In Vivo Proteome-Wide Measurement of Protein Kinetics Using Metabolic Labeling.

Control of biosynthetic and catabolic rates of polymers, including proteins, stands at the center of phenotype, physiologic adaptation, and disease pathogenesis. Advances in stable isotope-labeling concepts and mass spectrometric instrumentation now allow accurate in vivo measurement of protein synthesis and turnover rates, both for targeted proteins and for unbiased screening across the proteome. We describe here the underlying principles and operational protocols for measuring protein dynamics, focusing on metabolic labeling with (2)H2O (heavy water) combined with tandem mass spectrometric analysis of mass isotopomer abundances in trypsin-generated peptides. The core principles of combinatorial analysis (mass isotopomer distribution analysis or MIDA) are reviewed in detail, including practical advantages, limitations, and technical procedures to ensure optimal kinetic results. Technical factors include heavy water labeling protocols, optimal duration of labeling, clean up and simplification of sample matrices, accurate quantitation of mass isotopomer abundances in peptides, criteria for adequacy of mass spectrometric abundance measurements, and calculation algorithms. Some applications are described, including the noninvasive "virtual biopsy" strategy for measuring molecular flux rates in tissues through measurements in body fluids. In addition, application of heavy water labeling to measure flux lipidomics is noted. In summary, the combination of stable isotope labeling, particularly from (2)H2O, with tandem mass spectrometric analysis of mass isotopomer abundances in peptides, provides a powerful approach for characterizing the dynamics of proteins across the global proteome. Many applications in research and clinical medicine have been achieved and many others can be envisioned.

Highlighting the tricarboxylic acid cycle: Liquid and gas chromatography–mass spectrometry analyses of 13C-labeled organic acids

The tricarboxylic acid (TCA) cycle is involved in the complete oxidation of organic acids to carbon dioxide in aerobic cells. It not only uses the acetyl-CoA derived from glycolysis but also uses breakdown products of proteins, fatty acids, and nucleic acids. Therefore, the TCA cycle involves numerous carbon fluxes through central metabolism to produce reductant power and transfer the generated electrons to the aerobic electron transport system where energy is formed by oxidative phosphorylation. Although the TCA cycle plays a crucial role in aerobic organisms and tissues, the lack of direct isotopic labeling information in its intermediates (organic acids) makes the quantification of its metabolic fluxes rather approximate. This is the major technical gap that this study intended to fill. In this work, we established and validated liquid and gas chromatography–mass spectrometry methods to determine 13C labeling in organic acids involved in the TCA cycle using scheduled multiple reaction monitoring and single ion monitoring modes, respectively. Labeled samples were generated using maize embryos cultured with [13C]glucose or [13C]glutamine. Once steady-state labeling was reached, 13C-labeled organic acids were extracted and purified. When applying our mass spectrometric methods to those extracts, mass isotopomer abundances of seven major organic acids were successfully determined.

The Effect of Long Term Calorie Restriction on in Vivo Hepatic Proteostatis: A Novel Combination of Dynamic and Quantitative Proteomics

Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden.

Metabolic turnover analysis by a combination of in vivo13C-labelling from 13CO2 and metabolic profiling with CE-MS/MS reveals rate-limiting steps of the C3 photosynthetic pathway in Nicotiana tabacum leaves

Understanding of the control of metabolic pathways in plants requires direct measurement of the metabolic turnover rate. Sugar phosphate metabolism, including the Calvin cycle, is the primary pathway in C3 photosynthesis, the dynamic status of which has not been assessed quantitatively in the leaves of higher plants. Since the flux of photosynthetic carbon metabolism is affected by the CO2 fixation rate in leaves, a novel in vivo 13C-labelling system was developed with 13CO2 for the kinetic determination of metabolic turnover that was the time-course of the 13C-labelling ratio in each metabolite. The system is equipped with a gas-exchange chamber that enables real-time monitoring of the CO2 fixation rate and a freeze-clamp that excises a labelled leaf concurrently with quenching the metabolic reactions by liquid nitrogen within the photosynthesis chamber. Kinetic measurements were performed by detecting mass isotopomer abundance with capillary electrophoresis-tandem mass spectrometry. The multiple reaction monitoring method was optimized for the determination of each compound for sensitive detection because the amount of some sugar phosphates in plant cells is extremely small. Our analytical system enabled the in vivo turnover of sugar phosphates to be monitored in fresh tobacco (Nicotiana tabacum) leaves, which revealed that the turnover rate of glucose-1-phosphate (G1P) was significantly lower than that of other sugar phosphates, including glucose-6-phosphate (G6P). The pool size of G1P is 12 times lower than that of G6P. These results indicate that the conversion of G6P to G1P is one of the rate-limiting steps in the sugar phosphate pathway.

Effects of modified alternate-day fasting regimens on adipocyte size, triglyceride metabolism, and plasma adiponectin levels in mice

Calorie restriction (CR) affects adipocyte function and reduces body weight. However, the effects of alternate-day fasting (ADF) on adipose biology remain unclear. This study examined the effects of ADF and modified ADF regimens on adipocyte size, triglyceride (TG) metabolism, and adiponectin levels in relation to changes in body weight and adipose mass. Twenty-four male C57BL/6J mice were randomized for 4 weeks among 1) ADF-25% (25% CR on fast day, ad libitum on alternate day), 2) ADF-50% (50% CR on fast day), 3) ADF-100% (100% CR on fast day), and 4) control (ad libitum). The body weight of ADF-100% mice was lower than that of the other groups (P < 0.005) after treatment. Adipose tissue weights did not change. Inguinal and epididymal fat cells were 35-50% smaller (P < 0.01) than those of controls in ADF-50% and ADF-100% animals after treatment. Net lipolysis was augmented (P < 0.05) in ADF-100% mice, and the contribution from glyceroneogenesis to alpha-glycerol phosphate increased in ADF-50% and ADF-100% mice, whereas fractional and absolute de novo lipogenesis also increased in ADF-50% and ADF-100% animals, consistent with an alternating feast-fast milieu. Plasma adiponectin levels were not affected. In summary, modified ADF (ADF-50%) and complete ADF (ADF-100%) regimens modulate adipocyte function, despite there being no change in body weight or adipose tissue weight in the former group.

Physiologic and Pharmacologic Factors Influencing GlyceroneogenicContribution to Triacylglyceride Glycerol Measured by Mass IsotopomerDistribution Analysis

An imbalance between triacylglycerol synthesis and breakdown is necessary for the development of obesity. The direct precursor for triacylglycerol biosynthesis is alpha-glycerol phosphate, which can have glycolytic and glyceroneogenic origins. We present a technique for determining the relative glyceroneogenic contribution to triacylglyceride glycerol by labeling the glycerol moiety with 2H2O. The number of hydrogen atoms (n) incorporated from H2O into C-H bonds reflects the metabolic source of alpha-glycerol phosphate and can be calculated by combinatorial analysis of the distribution of mass isotopomers in triacylglyceride glycerol. Three physiological settings with potential effects on glyceroneogenesis and glycolysis were studied in rodents. Adipose tissue acylglyceride glycerol in mice fed a low carbohydrate diet had significantly higher values of n than in mice fed a high carbohydrate diet, suggesting an increased contribution from glyceroneogenesis of from 17 to 50% on the low carbohydrate diet. Similarly, mice administered rosiglitazone had a significant relative increase in glyceroneogenesis (from 17 to 53%), indicated by an increase in adipose acylglyceride glycerol n. Fructose infusion in overnight fasted rats rapidly lowered plasma triacylglyceride glycerol n, reflecting a decreased contribution from glyceroneogenesis (from 66 to 34%) presumably because of increased glycolytic input. In conclusion, we demonstrate that the number of C-H atoms derived from cellular H2O in triacylglyceride glycerol is an informative indicator of alpha-glycerol phosphate origin and, ultimately, triacylglycerol metabolism. Under certain physiological conditions, glyceroneogenesis can be up-regulated in adipose (e.g. low carbohydrate diet) or down-regulated in liver (e.g. fructose infusion). Additionally, stimulation of glyceroneogenesis by rosiglitazone in adipose tissue may be an important factor in the antilipolytic actions of thiazolidinediones.

Measurement of Age-Related Changes in Bone Matrix Using2H2O Labeling

Age-related changes in bone metabolism are well established by biochemical markers of bone matrix in serum and urine, but analysis of the residual bone matrix, which is still turning over, has not been investigated. In the present study, we measured in vivo rates of bone protein synthesis using a precursor-product method based on the exchange of ²H from ²H₂O into amino acids. Four percent ²H₂O was administered to mice in drinking water after intraperitonial (i.p) bolus injection of 99.9% ²H₂O. Mice were divided into the two groups: growing young mice were administered 4% ²H₂O for 12 weeks after an i.p bolus injection at 5 week of age, whereas weight stable adult mice started drinking 4% ²H₂O 8 weeks later than the growing group and continued 4% ²H₂O drinking for 8 weeks. Mass isotopomer abundance in alanine from bone protein was analyzed by gas chromatography/mass spectrometry. Body ²H₂O enrichments were in the range of 1.88~2.41% over the labeling period. The fractional synthesis rates (ks) of bone protein were 2.000±0.071%/d for growing mice and 0.243±0.014%/d for adult mice. These results demonstrate that the bone protein synthesis rate decreases with age and present direct evidence of age-related changes in bone protein synthesis.

Measuring synthesis rates of muscle creatine kinase and myosin with stable isotopes and mass spectrometry

We investigated a novel strategy for measuring the synthesis rate of proteins in skeletal and cardiac muscle. Mass isotopomer distribution analysis allows measurement of the isotopic enrichment of the true biosynthetic precursor for proteins (tRNA-amino acids), but cannot easily be applied to slow turnover muscle proteins due to insufficient isotope incorporation into multiply labeled species. Using a rapid turnover protein from the same tissue, however, might reveal tRNA-amino acid enrichment. We tested this strategy in rats on muscle creatine kinase (CK). A trypsinization peptide (3647 u) containing 5 leucine repeats was identified by computer-simulated digestion of CK and then isolated from trypsin hydrolysates. Mass isotopomer abundances were determined by electrospray ionization-magnetic sector-mass spectrometry after in vivo administration of [ 2 H 3 ]leucine. Myosin heavy chain was also isolated and hydrolyzed to free amino acids. Muscle tRNA-amino acids were well labeled, by direct measurement. Enrichments of M +1 and M +2 mass isotopomers in the CK-peptide were measurable but low (consistent with a CK half-life of 3–10 days). Incorporation into skeletal muscle myosin indicated a half-life of 54 days. In conclusion, the general strategy of measuring protein kinetics by quantifying mass isotopomer abundances of mid-sized peptides from protein hydrolysates is effective, but CK does not turn over rapidly in muscle, contrary to previous reports. Identification of a rapid turnover muscle protein would be useful for this purpose.

Elimination of the concentration dependence in mass isotopomer abundance mass spectrometry of methyl palmitate using metastable atom bombardment

An important problem in mass isotopomer abundance mass spectrometry (MIAMS) is the dependence of measured mass isotopomer abundances on sample concentration. We have evaluated the role of ionization energy on mass isotopomer abundance ratios of methyl palmitate as a function of sample concentration. Ionization energy was varied using electron impact ionization (EI) and metastable atom bombardment (MAB). The latter generates a beam of metastable species capable of ionizing analyte molecules by Penning ionization. We observed that ionization of methyl palmitate by EI (70 eV) showed the greatest molecular ion fragmentation and also showed the greatest dependence of relative isotopomer abundance ratios on sample concentration. Ionization using the 3 P 2 and 3 P 0 states of metastable krypton (9.92 and 10.56 eV, respectively) resulted in almost no molecular ion fragmentation, and the isotopomer abundances quantified were essentially independent of sample concentration. Ionization using the 3 P 2 and 3 P 0 states of metastable argon (11.55 and 11.72 eV, respectively) showed molecular ion fragmentation intermediate between that of EI and MAB(Kr) and showed an isotopomer concentration dependence which was less severe than that observed with EI but more severe than that observed with MAB(Kr). The observed decrease in the dependence of isotopomer abundance on sample concentration with a decrease in molecular ion fragmentation is consistent with the hypothesis that proton transfer from a fragment cation to a neutral molecule is the gas phase reaction mechanism responsible for the concentration dependence. Alternative explanations, e.g., hydrogen abstraction from a neutral molecule to a molecular cation, is not supported by these results. Moreover, the MAB ionization technique shows potential for eliminating one source of error in MIAMS measurements of methyl palmitate, in particular, and of fatty acids methyl esters, in general.

Deuterium in vivo labelling of cytokinins in Arabidopsis thaliana analysed by capillary liquid chromatography/frit-fast atom bombardment mass spectrometry.

A method was developed for analysing the biosynthetic rate of the cytokinin class of plant hormones. Transgenic, cytokinin-overproducing Arabidopsis thaliana plants were incubated in liquid culture media enriched with 30% deuterium oxide, and incorporation into the different parts of the cytokinin molecule was analysed by capillary liquid chromatography/frit-fast atom bombardment mass spectrometry after precolumn propionylation. The sugar moieties of the cytokinins generally showed a high and independent incorporation, so the analysis in this study focused on the cytokinin base moieties. It was observed that during a 24 h incubation period almost all labelling was incorporated into the side-chain, rather than the adenine moiety. The incorporation dynamics of isopentenyladenosine-5'-monophosphate, zeatinriboside-5'-monophosphate (ZRMP) and zeatin-9-glucoside were investigated through analysis of the cytokinin base fragments in high-resolution selective ion monitoring mode. Using a fractional synthetic rate approach, the biosynthetic rate of ZRMP was determined to be 18 ng h(-1) g(-1) fresh weight, giving a turnover time of 25 h. A method for the mass isotopomer abundance analysis of the cytokinins in the zeatin family, based on selective reaction monitoring, was also developed to gain further sensitivity. Use of this technique showed that there was a higher level of enrichment in zeatin nucleotide than in the corresponding nucleoside, in agreement with the hypothesis that cytokinin nucleotides are primary products in this pathway.

Molecular ion fragmentation and its effects on mass isotopomer abundances of fatty acid methyl esters ionized by electron impact

We have analyzed the isotopomer abundance ratios of an equimolar mixture of nine fatty acid methyl esters (decanoate, undecanoate, laurate, tridecanoate, myristate, pentadecanoate, palmitate, heptadecanoate, and stearate) by selected-ion monitoring gas chromatography/electron impact/mass spectrometry (GC/EI/MS). The abundance of the second lowest m/ z isotopomer ( I M 1 ) increased disproportionately compared with the abundance of the lowest m/ z isotopomer ( I M 0 ) as a function of: (1) increasing sample size; (2) decreasing repeller voltage; and (3) decreasing alkyl chain length. We also compared the abundance of the third lowest m/ z isotopomer ( I M 2 ) and the abundance of the second lowest m/ z isotopomer ( I M 1 ) of methyl palmitate and [4,4- 2H 2]methyl palmitate. We observed that the I M 2 / I M 1 for methyl palmitate was significantly lower than I M 2 / I M 1 for [4,4- 2H 2]methyl palmitate. From these results, as well as a consideration of basic principles of ion chemistry and ion physics, we conclude that gas-phase chemistry, specifically proton (or deuteron) transfer from fragment ions to molecules, is a major contributor to the sample size dependence observed in mass isotopomer abundance measurements of fatty acid methyl esters ionized by EI. Our results and analysis do not support hydrogen abstraction as the reaction mechanism. In addition, we calculate that rearranged molecular ions are unlikely to contribute significantly to intermolecular proton transfer because of their relatively brief lifetime. We also discuss alternative analytical techniques which might improve the precision and accuracy of isotopomer measurements by reducing molecular ion fragmentation.

Measuring Protein Synthesis by Mass Isotopomer Distribution Analysis (MIDA)

The measurement of protein kinetics by isotopic techniques has been hindered by the long-standing difficulty of accurately measuring the isotope content of the biosynthetic precursor pool (aminoacyl-tRNA in tissues). Mass isotopomer distribution analysis (MIDA) is a stable isotope-mass spectrometric (MS) technique for measuring biosynthetic precursor enrichments from measurements on a polymeric product, based on combinatorial probabilities of labeled and unlabeled monomeric subunits. Proteins contain complex isotopomer patterns as a result of their relatively high molecular mass, however, so that resolution of the individual mass isotopomers in the polymeric product (an analytic requirement for MIDA) is technically difficult. An approach for measuring protein synthesis by MIDA is described and tested here: First,in vitro,using a synthetic peptide present in human serum albumin; and then,in vivo,for albumin synthetic rate in rats. A peptide contained in human serum albumin (SVVLLLR) and theoretically recoverable from trypsin/chymotrypsin proteolysis was synthesized by solid-phase peptide synthesis using known mixtures of natural abundance and [5,5,5-2H3]leucine. Additionally, enriched and natural abundance peptides were mixedin vitroto simulatein vivobiosynthesis and to address problems of instrument accuracy, precision, and data management. The mass isotopomer patterns of the synthetic peptides were analyzed using electrospray ionization (ESI) with both magnetic sector and quadrupole mass analyzers. The resolution of the magnetic sector was superior to that of the quadrupole instrument, but accuracy and precision in the measurement of mass isotopomer abundances and kinetic parameters were comparable and both gave values close to those predicted. Next, rats were infused with [5,5,5-2H3]leucine intravenously, and a leucine-rich peptide was isolated and purified from trypsin-digested rat serum albumin (RHPDYSVSLLLR, 1456 Da) and then analyzed by ESI-MS using a magnetic sector instrument. Precursor pool enrichments and fractional synthetic rates (0.45 ± 0.03 day−1,t1/2= 1.53 ± 0.09 days) were calculated. Biosynthetic rates of rat serum albumin were congruent with previously published values. In summary, measurement of protein synthesis and precursor pool enrichments by MIDA is technically feasible and practicalin vivousing proteolytically derived peptides and ESI-MS analysis.

Gas-phase acid-base chemistry and its effects on mass isotopomer abundance measurements of biomolecular ions

Various parameters which affect mass isotopomer abundance measurements of derivatives of palmitic acid ionized by electron ionization (EI) and electron-capture negative chemical ionization (ECNCI) were tested on a sector-field double-focusing mass spectrometer. Results on methyl palmitate ionized by EI are as follows: (i) sample size had a significant effect on mass isotopomer abundance ratios (MIARs); (ii) the electron multiplier gain of the detector also had an effect on MIARs; and (iii) ion scattering by ion–neutral collisions in the mass analyzer did not appear to have any significant effect on MIARs (under standard analysis conditions). However, ‘reagent’ gas pressure (methane) had a significant effect on MIARs of pentafluorylbenzyl palmitate ionized by ECNCI. It was concluded that there are two compensatory effects which alter MIARs of methyl palmitate ionized by EI: (i) gas-phase acid–base chemistry in the source (specifically, proton transfer between fragment cations and neutral molecules); and (ii) detector non-linearities, specifically, underestimation of less abundant isotopomers due to their signal disproportionately falling within the signal-to-noise ratio level of the electron multiplier. Gas-phase chemistry is the dominant cause of inaccuracy in MIAR measurements for large sample sizes, while detector non-linearity is the dominant cause of inaccuracy in MIAR measurements at small sample sizes. However, in a narrow intermediate range of sample size, these two effects balance each other and result in MIARs which are ‘acceptable’ when compared with the known MIAR values. It is emphasized that these two effects are present regardless of the type of mass analyzer used, e.g. quadrupole, sector-field. Improvements in the accuracy of MIAR measurements will require developments in mass spectrometry aimed at eliminating each of the contributory effects. © 1998 John Wiley & Sons, Ltd.

Correction of 13C mass isotopomer distributions for natural stable isotope abundance.

Metabolism of singly or multiply 13C-labeled substrates leads to the production of molecules that contain 13C atoms at various positions. Molecules differing only in the number of isotopic atoms incorporated are referred to as mass isotopomers. The distribution of mass isotopomers of many molecules can be measured by gas chromatography/ mass spectrometry after chemical derivatization. Quantification of metabolite mass isotopomer abundance resulting from biological processes necessitates correction of the measured mass isotopomer distribution of the derivatized metabolite for contributions due to naturally occurring isotopes of its elements. This correction must take into account differences in the relative natural abundance distribution of each mass isotopomer (skewing). An IBM-compatible computer program was developed which (i) calculates the natural abundance mass isotopomer distribution of unlabeled and labeled standards given the molecular formula of the derivatized molecule or fragment ion, and (ii) calculates the natural abundance mass isotopomer distribution of the singly and multiply labeled molecule or fragment via non-linear fitting to the measured mass isotopomer distribution of the unlabeled molecule or fragment. The output of this program is used to correct measured mass isotopomer distributions for contributions from natural isotope abundances and to verify measured values for theoretical consistency. Differences between predicted and measured unlabeled and 13C-labeled isotopomer distributions for hydroxamate di-t-butyl-dimethylsilyl (di-TBDMS) derivatized pyruvate were measured. The program was applied to the mass isotopomer distribution of glucose labeled from [U-13C3]glycerol and of fatty acids labeled from [U-13C6]glucose and either [2-13C2] acetate or [U-13C2]acetate. In some of these cases, the measured mass isotopomer distributions corrected by the program were different from those corrected by the classical technique. Implications of these differences including those on the calculation of glucose production due to gluconeogenesis in isolated perfused rat liver are discussed.