Measuring protein synthesis using metabolic ²H labeling, high-resolution mass spectrometry, and an algorithm

We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O.     

Equilibration of H labeling between body water and free amino acids: Enabling studies of proteome synthesis

Protein synthesis can be estimated by measuring the incorporation of a labeled amino acid into a proteolytic peptide. Although prelabeled amino acids are typically administered, recent studies have tested ²H₂O; the assumption is that there is rapid equilibration of ²H (in body water) with the carbon-bound hydrogens of amino acids before those amino acids are incorporated into a protein(s). We have determined the temporal changes in ²H labeling of body water and amino acids which should build confidence in ²H₂O-based studies of protein synthesis when one aims to measure the ²H labeling of proteolytic peptides.     

Measuring Proteome Dynamics in Vivo: AS EASY AS ADDING WATER?

Proteomics investigations typically yield information regarding static gene expression profiles. The central issues that limit the study of proteome dynamics include how to (i) administer a labeled amino acid in vivo, (ii) measure the isotopic labeling of a protein(s) (which may be low), and (iii) reliably interpret the precursor/product labeling relationships. In this study, we demonstrate the potential of quantifying proteome dynamics by coupling the administration of stable isotopes with mass spectrometric assays. Although the direct administration of a labeled amino acid(s) is typically used to measure protein synthesis, we explain the application of labeled water, comparing ²H₂O versus H₂¹⁸O for measuring albumin biosynthesis in vivo. This application emphasizes two distinct advantages of using labeled water over a labeled amino acid(s). First, in long term studies (e.g. days or weeks), it is not practical to continuously administer a labeled amino acid(s); however, in the presence of labeled water, organisms will generate labeled amino acids. Second, to calculate rates of protein synthesis in short term studies (e.g. hours), one must utilize a precursor/product labeling ratio; when using labeled water it is possible to reliably identify and easily measure the precursor labeling (i.e. water). We demonstrate that labeled water permits studies of protein synthesis (e.g. albumin synthesis in mice) during metabolic “steady-state” or “non-steady-state” conditions, i.e. integrating transitions between the fed and fasted state or during an acute perturbation (e.g. following a meal), respectively. We expect that the use of labeled water is applicable to wide scale investigations of proteome dynamics and can therein be used to obtain a functional image of gene expression in vivo.Proteomics investigations typically yield information regarding static gene expression profiles; i.e. current “state-of-the-art” research programs lack measurements of proteome dynamics (1–3). This deficiency is unfortunate because the ability to measure rates of protein synthesis and breakdown will likely facilitate the identification of biomarkers of disease and yield novel insight regarding underlying homeostatic abnormalities (3, 4). For example, by measuring the concentration of circulating aminotransferase and the synthesis/secretion of albumin, one might be able to determine the degree of liver damage and assess whether hepatic function is compromised, respectively (5). Also, it should be possible to determine the influence of specific factors on the regulation of protein synthesis; e.g. does a therapeutic agent stimulate insulin biosynthesis?Classic studies of protein biosynthesis have measured the incorporation of a labeled amino acid(s) into a protein(s) of interest and estimated a synthesis rate by using a “precursor/product labeling ratio” (6). Because modern proteomics technologies can rapidly separate and quantify individual proteins from complex mixtures, investigators have started to exploit the use of stable isotope tracers in mass spectrometry-based studies of proteome kinetics. However, the ability to study protein dynamics in vivo presents unique challenges (3, 4, 7–13); e.g. how does one (i) administer an isotope (typically a labeled amino acid) over a prolonged period and (ii) determine the true precursor labeling (because the amino acid will be rapidly turned over and its labeling will be diluted)? We have demonstrated how to quantify protein synthesis using ²H₂O in vivo (10, 11); the advantages are that the tracer can be given orally, body water is a homogeneous pool with a relatively slow turnover, and the organism will continuously generate ²H-labeled amino acids (consequently one can study free living subjects, including humans (9, 11, 14)). The assumption of the method is that the equilibration between ²H in body water and a free amino acid(s) is faster than the rate of incorporation of an amino acid(s) into a newly made protein(s); preferably, the labeling of a free amino acid(s) should remain constant regardless of the metabolic status. We have validated that assumption by measuring the time-dependent labeling of alanine in vivo during the administration of ²H₂O and by measuring the incorporation of ²H-labeled alanine into plasma albumin and total tissue proteins using gas chromatography-mass spectrometry methods (10, 11, 15). Subsequent reports support our observations (12, 13).In this study, we demonstrate (as a model example) the application of our ²H₂O-based approach for measuring albumin biosynthesis in vivo in mice during long term and short term investigations. Namely, we recently demonstrated how to obtain relatively precise measurements of mass isotopomer profiles of peptides and other relatively large molecules by developing a novel approach for integrating the data (16, 17). Our method allowed us to detect shifts in the isotope distribution profile of albumin-derived peptides from mice given ²H₂O (17). In the current report, parallel studies examined the use of H₂¹⁸O because it offers potential advantages over ²H₂O, especially during acute studies that involve perturbations such as consumption of a meal. For example, the cleavage of a protein will immediately add a labeled oxygen atom into the carboxyl group of a free amino acid; resonance effects will distribute the label over both carboxyl oxygens. Although repeated cleavage is required to achieve maximal labeling of both oxygens, cleavage of tRNA-bound amino acids will also contribute to the labeling of the carboxyl oxygen (18–21). The synthesis of a new protein(s) then results in the stable incorporation of ¹⁸O into the peptide bond; indeed, the oxygen in peptide bonds accounts for a majority of the total oxygen in a protein (18, 19), making it potentially easier to describe precursor/product labeling relationships (6). Finally, during the development of this work pitfalls were identified; thus we discuss strategies to circumvent potential problems.     

Headspace analyses of H labeling of acetone: Enabling studies of fatty acid oxidation in vivo

We demonstrate that one can measure low levels of ²H labeling (e.g., <0.025% excess ²H) by exchanging hydrogen (deuterium) in water with acetone and subjecting samples to gas chromatography–pyrolysis–isotope ratio mass spectrometry. This analytical method circumvents the need to use typical off-line reduction methods that convert water to hydrogen gas prior to isotope ratio mass spectrometry or the need to purchase extra peripheral devices that would permit the direct analysis of water labeling. This method enables routine measurements of fatty acid oxidation in rodents; that is, one administers a ²H-labeled fatty acid(s) and then quantifies the production of ²H-labeled water.     

Improvement of chemical analysis of antibiotics: XXIII. Identification of residual tetracyclines in bovine tissues by electrospray high-performance liquid chromatography–tandem mass spectrometry

To reliably identify the residual tetracycline antibiotics (TCs), oxytetracycline (OTC), tetracycline, chlortetracycline (CTC) and doxycycline (DC), in bovine tissues, we have established a confirmation method using electrospray ionization liquid chromatography–tandem mass spectrometry (ESI LC–MS–MS) with daughter ion scan. All TCs gave [M+H−NH₃]⁺ and [M+H−NH₃−H₂O]⁺ as the product ions, except for DC when [M+H]⁺ was selected as the precursor ion. The combination of C₁₈ cartridge clean-up and the present ESI LC–MS–MS method can reliably identify TCs fortified at a concentration of 0.1 ppm in bovine tissues, including liver, kidney and muscle, and has been successfully applied to the identification of residual OTC in bovine liver and residual CTC in bovine muscle samples previously found at concentrations of 0.58 ppm and 0.38 ppm by LC, respectively.     

Topograph,a Software Platform for Precursor Enrichment Corrected Global Protein Turnover Measurements

Defects in protein turnover have been implicated in a broad range of diseases, but current proteomics methods of measuring protein turnover are limited by the software tools available. Conventional methods require indirect approaches to differentiate newly synthesized protein when synthesized from partially labeled precursor pools. To address this, we have developed Topograph, a software platform which calculates the fraction of peptides that are from newly synthesized proteins and their turnover rates. A unique feature of Topograph is the ability to calculate amino acid precursor pool enrichment levels which allows for accurate calculations when the precursor pool is not fully labeled, and the approach used by Topograph is applicable regardless of the stable isotope label used. We validate the Topograph algorithms using data acquired from a mouse labeling experiment and demonstrate the influence that precursor pool corrections can have on protein turnover measurements.Methods of measuring protein synthesis and degradation using stable or radioactive isotope labels have existed for decades. The isotope label is introduced in the form of a labeled amino acid or amino acid precursor, and the incorporation or removal of that label from protein is used to estimate average protein turnover rates (1, 2). Historically, the amount of stable isotope label incorporated into a protein is measured by enriching for the protein (e.g. affinity chromatography, gel electrophoresis, and other biochemical methods), hydrolyzing the protein to amino acids, derivatizing the amino acids, and measuring the labeled amino acid by gas chromatography-mass spectrometry or gas chromatography-combustion-isotope ratio mass spectrometry (3, 4). More recently, proteomics methods have been developed that measure the labeled amino acid on the peptide level, eliminating the need for a protein enrichment step and enabling the monitoring of many proteins in a single experiment (5).Proteomics approaches to measuring protein turnover rates in mice have been accomplished by the introduction of a ¹⁵N stable isotope label. The labeled diets were created by supplementing a protein-free diet with a ¹⁵N enriched protein source. Price et al. (6) generated ¹⁵N-labeled protein from the alga, Spirulina platensis and Zhang et al. (7) introduced ¹⁵N-label in the form of lysate from the bacterium, Ralstonia eutropha. An advantage of using complete ¹⁵N labeling is the rapid incorporation of ¹⁵N and separation of isotope distributions between labeled and natural isotope abundance peptides, which reduces the need to deconvolute the two distributions. However, current methods require that the dietary protein content be derived from bacterial or alga lysate, a diet that is not normally fed to laboratory mice. As a result, measurements of protein turnover may not reflect conventional mouse model systems because of effects of diet on protein and amino acid metabolism. A more recent work by Claydon et al. (8) demonstrated a stable isotope labeling method by supplementing labeled valine into a standard mouse diet.The complex data generated from these analyses creates a data processing and analysis challenge; exemplified by recent software platforms that have been developed. Guan et al. (9) and Hoopmann et al. (10) demonstrated data analysis pipelines for ¹⁵N labeled SILAM and SILAC experiments. Here we describe the software platform, Topograph, we have developed for the analysis of liquid chromatography-tandem MS (LC-MS/MS) data from samples with isotopic labels. Topograph is able to deconvolute the complex spectra that may result from overlapping isotope distributions, regardless of the isotope label used. More uniquely, Topograph is able to calculate the relative isotope abundance (RIA)¹ of the amino acid precursor pool, which is necessary to correctly determine the amount of newly synthesized peptide and to subsequently calculate peptide and protein turnover rates.     

A novel approach for assessing protein synthesis in channel catfish, Ictalurus punctatus

A comprehensive understanding of animal growth requires adequate knowledge of protein synthesis (PS), which in fish, has traditionally been determined by the flooding dose method. However, this procedure is limited to short-term assessments and may not accurately describe fish growth over extended periods of time. Since deuterium oxide (²H₂O) has been used to non-invasively quantify PS in mammals over short- and long-term periods, we aimed at determining if ²H₂O could also be used to measure PS in channel catfish. Fish were stocked in a 40-L aquarium with ~ 4% ²H₂O and sampled at 4, 8 and 24 h (n = 6 at each time period) to determine ²H-labeling of body water (plasma), as well as protein-free and protein-bound ²H-labeled alanine. The labeling of body water reflected that of aquarium water and the labeling of protein-free alanine remained constant over 24 h and was ~ 3.8 times greater than that of body water. By measuring ²H-labeled alanine incorporation after 24 h of ²H₂O exposure we were able to calculate a rate of PS: 0.04 ± 0.01% h^− 1. These results demonstrate that PS in fish can be effectively measured using ²H₂O and, because this method yields integrative measures of PS, is relatively inexpensive and accounts for perturbations such as feeding, it is a novel and practical assessment option.     

Combining recombinant ribonuclease U2 and protein phosphatase for RNA modification mapping by liquid chromatography–mass spectrometry

Ribonuclease (RNase) mapping of modified nucleosides onto RNA sequences is limited by RNase availability. A codon-optimized gene for RNase U2, a purine selective RNase with preference for adenosine, has been designed for overexpression using Escherichia coli as the host. Optimal expression conditions were identified enabling generation of milligram-scale quantities of active RNase U2. RNase U2 digestion products were found to terminate in both 2′,3′-cyclic phosphates and 3′-linear phosphates. To generate a homogeneous 3′-linear phosphate set of products, an enzymatic approach was investigated. Bacteriophage lambda protein phosphatase was identified as the optimal enzyme for hydrolyzing cyclic phosphates from RNase U2 products. The compatibility of this enzymatic approach with liquid chromatography–tandem mass spectrometry (LC–MS/MS) RNA modification mapping was then demonstrated. RNase U2 digestion followed by subsequent phosphatase treatment generated nearly 100% 3′-phosphate-containing products that could be characterized by LC–MS/MS. In addition, bacteriophage lambda protein phosphatase can be used to introduce ¹⁸O labels within the 3′-phosphate of digestion products when incubated in the presence of H₂¹⁸O, allowing prior isotope labeling methods for mass spectrometry to include digestion products from RNase U2.     

Determination of muscle protein synthesis rates in fish using H2O and H NMR analysis of alanine

Following administration of deuterated water (²H₂O), the fractional synthetic rate (FSR) of a given endogenous protein can be estimated by ²H-enrichment quantification of its alanine residues. Currently, this is measured by mass spectrometry following a derivatization procedure. Muscle FSR was measured by ¹H/²H NMR analysis of alanine from seabass kept for 6 days in 5% ²H-enriched saltwater, following acid hydrolysis and amino acid isolation by cation-exchange chromatography of muscle tissue. The analysis is simple and robust, and provides precise measurements of excess alanine ²H-enrichment in the 0.1–0.4% range from 50 mmol of alanine recovered from muscle protein.     

Oxidized fatty acid analysis by charge-switch derivatization,selected reaction monitoring,and accurate mass quantitation

A highly sensitive, specific, and robust method for the analysis of oxidized metabolites of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was developed using charge-switch derivatization, liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI MS/MS) with selected reaction monitoring (SRM) and quantitation by high mass accuracy analysis of product ions, thereby minimizing interferences from contaminating ions. Charge-switch derivatization of LA, AA, and DHA metabolites with N-(4-aminomethylphenyl)-pyridinium resulted in a 10- to 30-fold increase in ionization efficiency. Improved quantitation was accompanied by decreased false positive interferences through accurate mass measurements of diagnostic product ions during SRM transitions by ratiometric comparisons with stable isotope internal standards. The limits of quantitation were between 0.05 and 6.0 pg, with a dynamic range of 3 to 4 orders of magnitude (correlation coefficient r² > 0.99). This approach was used to quantitate the levels of representative fatty acid metabolites from wild-type (WT) and iPLA₂γ^–/– mouse liver identifying the role of iPLA₂γ in hepatic lipid second messenger production. Collectively, these results demonstrate the utility of high mass accuracy product ion analysis in conjunction with charge-switch derivatization for the highly specific quantitation of diminutive amounts of LA, AA, and DHA metabolites in biologic systems.     

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.     

Can dimedone be used to study selenoproteins? An investigation into the reactivity of dimedone toward oxidized forms of selenocysteine

Dimedone is a widely used reagent to assess the redox state of cysteine‐containing proteins as it will alkylate sulfenic acid residues, but not sulfinic acid residues. While it has been reported that dimedone can label selenenic acid residues in selenoproteins, we investigated the stability, and reversibility of this label in a model peptide system. We also wondered whether dimedone could be used to detect seleninic acid residues. We used benzenesulfinic acid, benzeneseleninic acid, and model selenocysteine‐containing peptides to investigate possible reactions with dimedone. These peptides were incubated with H₂O₂ in the presence of dimedone and then the reactions were followed by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS). The native peptide, H‐PTVTGCUG‐OH (corresponding to the native amino acid sequence of the C‐terminus of mammalian thioredoxin reductase), could not be alkylated by dimedone, but could be carboxymethylated with iodoacetic acid. However the “mutant peptide,” H‐PTVTGAUG‐OH, could be labeled with dimedone at low concentrations of H₂O₂, but the reaction was reversible by addition of thiol. Due to the reversible nature of this alkylation, we conclude that dimedone is not a good reagent for detecting selenenic acids in selenoproteins. At high concentrations of H₂O₂, selenium was eliminated from the peptide and a dimeric form of dimedone could be detected using LCMS and ¹H NMR. The dimeric dimedone product forms as a result of a seleno‐Pummerer reaction with Sec‐seleninic acid. Overall our results show that the reaction of dimedone with oxidized cysteine residues is quite different from the same reaction with oxidized selenocysteine residues.     

Mass spectrometric analysis of N-carboxymethylamino acids as periodate oxidation derivatives of Amadori compounds application to glycosylated haemoglobin

Summary Periodate oxidation of free and protein-bound Amadori compounds formed by the condensation of reducing sugars with primary amino groups generates, on acid hydrolysis, N-carboxymethyl derivatives of amino acids. The analysis of these modified amino acids may be used to estimate both the extent and the site of protein glycosylation. The present study describes the use of gas chromatography-mass spectrometry (GC/MS) and gas chromatographytandem mass spectrometry (GC/MS/MS) for the identification of the various N-carboxymethylamino acids. Application of this approach to the quantitation of N-carboxymethylvaline and N -carboxymethyllysine resulting from the oxidation of glycosylated haemoglobin is presented.     

Identification of isomerization and racemization of aspartate in the Asp-Asp motifs of a therapeutic protein

A thermally stressed Fab molecule showed a significant increase of basic variants in imaged capillary isoelectric focusing (iCIEF) analysis. Mass analyses of the reduced protein found an increase in −18 Da species from both light chain and heavy chain. A tryptic peptide map identified two isoAsp-containing peptides, both containing Asp–Asp motifs and located in complementarity-determining regions (CDRs) of light chains and heavy chains, respectively. The approaches of hydrolyzing succinimide in H₂¹⁸O followed by tryptic digestion were used to label and identify the sites of isomerization. This method enabled identification of the isomerization site by comparing the MS/MS spectra of isomerized peptides with and without ¹⁸O incorporation. The light chain peptide L2 VTITCITSTDID₁₂DDMNWYQQKPGK underwent simultaneous isomerization and recemization at residue Asp-12 after thermal stress as evidenced by the coinjection of synthetic peptide L2 with l-Asp-12, l-isoAsp-12, d-Asp-12, and d-isoAsp-12, respectively. A thermal stress study of the synthetic peptide (l-)L2 showed that the isomerization and racemization did not occur, indicating that the Asp degradation in this Asp–Asp motif is more related to the protein conformation than the primary sequence. Another isomerization site was identified as Asp-24 in the heavy chain peptide H5 QAPGQGLEWMGWINTYTGETTYAD₂₄DFK. No other isomerizations were detected in CDR peptides containing either Asp–Ser or Asp–Thr motifs.     

Reproducibility of an HPLC-ESI-MS/MS method for the measurement of stable-isotope enrichment of in vivo-labeled muscle ATP synthase beta subunit

We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase β subunit (β-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, β-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple β-F1-ATPase peptides. There were three β-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to β-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R² = 0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the β-F1-ATPase_134-143 peptide. Measured stable-isotope enrichment was highly reproducible for the β-F1-ATPase_134-143 peptide (CV = 2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R² = 0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the β-F1-ATPase_134-143 peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle β-F1-ATPase based on the determination of the enrichment of the β-F1-ATPase_134-143 peptide.     

Comparison of gas chromatography–mass spectrometry and gas chromatography–combustion–isotope ratio mass spectrometry analysis for in vivo estimates of metabolic fluxes

Gas chromatography–mass spectrometry (GC–MS) was compared with gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for measurements of cholesterol ¹³C enrichment after infusion of labeled precursor ([¹³C_1,2]acetate). Paired results were significantly correlated, although GC–MS was less accurate than GC–C–IRMS for higher enrichments. Nevertheless, only GC–MS was able to provide information on isotopologue distribution, bringing new insights to lipid metabolism. Therefore, we assessed the isotopologue distribution of cholesterol in humans and dogs known to present contrasted cholesterol metabolic pathways. The labeled tracer incorporation was different in both species, highlighting the subsidiarity of GC–MS and GC–C–IRMS to analyze in vivo stable isotope studies.     

Stable isotope labeling by amino acids in cell culture-based liquid chromatography–mass spectrometry assay to measure microtubule dynamics in neuronal cell cultures

Microtubules (MTs) are highly dynamic polymers composed of α- and β-tubulin heterodimers. Dysregulation of MT dynamics in neurons may be a contributing factor in the progression of various neurodegenerative diseases. We developed a stable isotope labeling by amino acids in cell culture (SILAC)-based liquid chromatography–mass spectrometry (LC–MS) method to measure the fraction of [¹³C₆]leucine-labeled α-tubulin-derived surrogate peptides. Using this approach, we measured the time course of incorporation of [¹³C₆]leucine label into the MT and dimer pools isolated from cycling cells and rat primary hippocampal neurons. We found that the MT pool is in rapid equilibrium with the dimer pool in the cycling cells, consistent with rapid MT polymerization/depolymerization during cell proliferation. Conversely, in neurons, we found that labeling of the MT pool was rapid, whereas the dimer pool was delayed. These results suggest that newly synthesized α-tubulin is first incorporated into MTs or complexes that co-sediment with MTs and that appearance of labeled α-tubulin in the dimer pool may be a consequence of MT depolymerization or breakdown. Our results demonstrate that a SILAC-based approach can be used to measure MT dynamics and may have utility for exploring MT dysregulation in various models of neurodegenerative disease.     

Isotopomer enrichment assay for very short chain fatty acids and its metabolic applications

The present work illustrated an accurate GC/MS measurement for the low isotopomer enrichment assay of formic acid, acetic acid, propionic aicd, butyric acid, and pentanoic acid. The pentafluorobenzyl bromide derivatives of these very short chain fatty acids have high sensitivity of isotopoic enrichment due to their low natural isotopomer distribution in negative chemical ionization mass spectrometric mode. Pentafluorobenzyl bromide derivatization reaction was optimized in terms of pH, temperature, reaction time, and the amount of pentafluorobenzyl bromide versus sample. The precision, stability, and accuracy of this method for the isotopomer analysis were validated. This method was applied to measure the enrichments of formic acid, acetic acid, and propionic acid in the perfusate from rat liver exposed to Krebs–Ringer bicarbonate buffer only, 0–1 mM [3,4-¹³C₂]-4-hydroxynonanoate, and 0–2 mM [5,6,7-¹³C₃]heptanoate. The enrichments of acetic acid and propionic acid in the perfusate are comparable to the labeling pattern of acetyl-CoA and propionyl-CoA in the rat liver tissues. The enrichment of the acetic acid assay is much more sensitive and precise than the enrichment of acetyl-CoA by LC-MS/MS. The reversibility of propionyl-CoA from succinyl-CoA was confirmed by the low labeling of M1 and M2 of propionic acid from [5,6,7-¹³C₃]heptanoate perfusates.     

Measurement of N enrichment of glutamine and urea cycle amino acids derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using liquid chromatography–tandem quadrupole mass spectrometry

6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is an amino acid-specific derivatizing reagent that has been used for sensitive amino acid quantification by liquid chromatography–tandem quadrupole mass spectrometry (LC–MS/MS). In this study, we aimed to evaluate the ability of this method to measure the isotopic enrichment of amino acids and to determine the positional ¹⁵N enrichment of urea cycle amino acids (i.e., arginine, ornithine, and citrulline) and glutamine. The distribution of the M and M + 1 isotopomers of each natural AQC–amino acid was nearly identical to the theoretical distribution. The standard deviation of the (M + 1)/M ratio for each amino acid in repeated measurements was approximately 0.1%, and the ratios were stable regardless of the injected amounts. Linearity in the measurements of ¹⁵N enrichment was confirmed by measuring a series of ¹⁵N-labeled arginine standards. The positional ¹⁵N enrichment of urea cycle amino acids and glutamine was estimated from the isotopic distribution of unique fragment ions generated at different collision energies. This method was able to identify their positional ¹⁵N enrichment in the plasma of rats fed ¹⁵N-labeled glutamine. These results suggest the utility of LC–MS/MS detection of AQC–amino acids for the measurement of isotopic enrichment in ¹⁵N-labeled amino acids and indicate that this method is useful for the study of nitrogen metabolism in living organisms.     

Carbamoylated free amino acids in uremia: HOCl generates volatile protein modifying and cytotoxic oxidant species from N-carbamoyl-threonine but not threonine

N-carbamoylation is the non-enzymatic reaction of cyanate with amino groups. Due to urea-formed cyanate in uremic patients beside carbamoylated proteins also free amino acid carbamoylation has been detected, a modification which has been linked to disturbed protein synthesis as NH₂-derivatisation interferes with peptide bond formation. HOCl the product of the activated MPO/H₂O₂/Cl⁻ system is known to react with the NH₂-group of free amino acids to form chloramines which could exert some protective effect against protein modification and cytotoxicity induced by HOCl. As N-carbamoylation may inhibit formation of chloramines we have used N-carbamoyl-threonine as a model amino acid to study its ability to limit the reactivity of HOCl with proteins (LDL and human serum albumin) and cells (THP-1 monocytes and coronary artery endothelial cells). The data indicate that N-carbamoylation completely abolished the protein- and cell-protective effect of threonine against HOCl attack. In contrast to threonine the reaction of HOCl with carbamoyl-threonine resulted in the formation of volatile oxidant species with protein modifying and cytotoxic potential. The volatile lipophilic inorganic monochloramine (NH₂Cl) was identified as a breakdown product of this reaction.