Stable Isotope Labeling Of Amino Acids Research Articles

The role of carbohydrate drinks in preoperative nutrition: comment 2

We read with interest the review of Jones et al looking at the role of CHO drinks in perioperative nutrition for elective colorectal surgery. However, we have some concerns regarding the rigour of assessment of elements of the evidence base and the authors’ accuracy in presentation of this evidence. The largest randomised controlled trial referenced (and one of only two that looked at clinical outcomes in patients undergoing colorectal surgery in the review) is that of Mathur et al. 1 The review by Jones et al states that Mathur et al showed CHO supplements reduced length of hospital stay. This is at odds with Mathur’s own conclusion: ‘Preoperative CHO treatment did not improve postoperative fatigue or length of hospital stay after major abdominal surgery.’ 1 Furthermore, the work of Svanfeldt et al 2 is misquoted in this review, suggesting it showed that: ‘whole-body protein did not change in the high CHO group whereas it was more negative in the low CHO group after surgery…’ Svanfeldt et al investigated whole-body protein kinetics via a stable isotope labelled amino acid technique and not whole-body protein. This protein kinetic study looked at changes in protein balance before and after colorectal surgery. Whole-body protein balance was shown to be negative at a set point in time during the preoperative fast and again at a set point during the early postoperative period. The rate of loss of protein mass at this instant was faster in the group receiving low dose preoperative CHO than in the high dose group. To interpret this as meaning whole-body protein did not change in the perioperative period in the group receiving high dose CHO is erroneous. While Yuill et al have shown a reduced loss of muscle mass postoperatively in patients receiving CHO, 3 this has been in upper gastrointestinal (GI) surgery and not colorectal surgery. Other studies such as that by Mathur et al 1 do measure total body protein in lower GI surgery but have not as yet demonstrated that CHO can significantly attenuate the perioperative loss of total body protein. We agree that measures to minimise the stress response to surgery will benefit our patients. Evidence exists that supports the implementation of ERAS programmes, 4 most of which include preoperative CHO supplementation. However, it is important for us to present in context an accurate evidence base supporting each component of ERAS to maximise the benefit to the patient with the minimum number of steps in the process. The case for CHO perioperative drink is not settled and should not become established as dogma (which applies to any other step in the process) until it is proven.

Translational Research in Complementary and Alternative Medicine

Translational Research in Complementary and Alternative Medicine

Determination of very low stable isotope enrichments of [2H5]-phenylalanine in chicken liver using liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Stable isotope labeled amino acids are frequently used to examine nutritive effects on protein synthesis. This technique is characterized by tracing the incorporation of the label into newly synthesized proteins. In the present investigation, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the determination of very low enrichment of protein-bound l-[2H5]-phenylalanine ([2H5]-phe) in chicken liver. The LC–MS/MS measurements were carried out in positive atmospheric pressure chemical ionization (APCI) mode. Two mass transitions each for [2H5]-phe (171.1/125.1 and 171.1/106.1) and l-phenylalanine (phe) (166.1/91.1 and 166.1/93.1) were chosen for quantification and qualification. Due to the high excesses of phe, less sensitive transitions were chosen in the case of phe. The separation was carried out on a phenyl-hexyl column using 0.1% formic acid as eluent A and methanol as eluent B. The method was calibrated with calibration standard solutions in the range of 0.01–0.5 mole percent excess (MPE). Linear regression analysis led to coefficients of determination (r2) greater than 0.9995. The method was applied on liver samples from experiments investigating nutritive effects on tissue protein synthesis in broiler chickens. These samples were analyzed with a gas chromatography–mass spectrometry (GC–MS) method and reanalyzed with the developed LC–MS/MS method one year later. Compared to GC–MS, the main advantages of the LC–MS/MS method are its higher selectivity as well as the elimination of the need to convert and derivatize the samples prior to measuring.

Stable Isotope Labeling with Amino Acids in Drosophila for Quantifying Proteins and Modifications

Drosophila melanogaster is a common animal model for genetics studies, and quantitative proteomics studies of the fly are emerging. Here, we present in detail the development of a procedure to incorporate stable isotope-labeled amino acids into the fly proteome. In the method of stable isotope labeling with amino acids in Drosophila melanogaster (SILAC fly), flies were fed with SILAC-labeled yeast grown with modified media, enabling near complete labeling in a single generation. Biological variation in the proteome among individual flies was evaluated in a series of null experiments. We further applied the SILAC fly method to profile proteins from a model of fragile X syndrome, the most common cause of inherited mental retardation in human. The analysis identified a number of altered proteins in the disease model, including actin-binding protein profilin and microtubulin-associated protein futsch. The change of both proteins was validated by immunoblotting analysis. Moreover, we extended the SILAC fly strategy to study the dynamics of protein ubiquitination during the fly life span (from day 1 to day 30), by measuring the level of ubiquitin along with two major polyubiquitin chains (K48 and K63 linkages). The results show that the abundance of protein ubiquitination and the two major linkages do not change significantly within the measured age range. Together, the data demonstrate the application of the SILAC principle in D. melanogaster, facilitating the integration of powerful fly genomics with emerging proteomics.

Characterization of Membrane-shed Microvesicles from Cytokine-stimulated β-Cells Using Proteomics Strategies

Microparticles and exosomes are two of the most well characterized membrane-derived microvesicles released either directly from the plasma membrane or released through the fusion of intracellular multivesicular bodies with the plasma membrane, respectively. They are thought to be involved in many significant biological processes such as cell to cell communication, rescue from apoptosis, and immunological responses. Here we report for the first time a quantitative study of proteins from β-cell-derived microvesicles generated after cytokine induced apoptosis using stable isotope labeled amino acids in cell culture combined with mass spectrometry. We identified and quantified a large number of β-cell-specific proteins and proteins previously described in microvesicles from other cell types in addition to new proteins located to these vesicles. In addition, we quantified specific sites of protein phosphorylation and N-linked sialylation in proteins associated with microvesicles from β-cells. Using pathway analysis software, we were able to map the most distinctive changes between microvesicles generated during growth and after cytokine stimulation to several cell death and cell signaling molecules including tumor necrosis factor receptor superfamily member 1A, tumor necrosis factor, α-induced protein 3, tumor necrosis factor-interacting kinase receptor-interacting serine-threonine kinase 1, and intercellular adhesion molecule 1.

Protein turnover: Measurement of proteome dynamics by whole animal metabolic labelling with stable isotope labelled amino acids

The measurement of protein turnover in tissues of intact animals is obtained by whole animal dynamic labelling studies, requiring dietary administration of precursor label. It is difficult to obtain full labelling of precursor amino acids in the diet and if partial labelling is used, calculation of the rate of turnover of each protein requires knowledge of the precursor relative isotope abundance (RIA). We describe an approach to dynamic labelling of proteins in the mouse with a commercial diet supplemented with a pure, deuterated essential amino acid. The pattern of isotopomer labelling can be used to recover the precursor RIA, and sampling of urinary secreted proteins can monitor the development of liver precursor RIA non-invasively. Time-series analysis of the labelling trajectories for individual proteins allows accurate determination of the first order rate constant for degradation. The acquisition of this parameter over multiple proteins permits turnover profiling of cellular proteins and comparisons of different tissues. The median rate of degradation of muscle protein is considerably lower than liver or kidney, with heart occupying an intermediate position.

A proteomics strategy for determining the synthesis and degradation rates of individual proteins in fish

In order to study the protein dynamics in the tissues of fish we have developed a proteomics-based strategy to determine the rates of synthesis and degradation of individual proteins. We have demonstrated the feasibility of this approach by measuring the turnover of multiple isoforms of parvalbumin (β1-7) in the skeletal muscle of common carp (Cyprinus carpio). A stable isotope-labelled amino acid ([(2)H(7)] l-leucine) was administered to the carp via the diet and its incorporation into the isoforms of parvalbumin in muscle over time was monitored by LC-MS analysis of signature peptides. The relative isotope abundance was calculated and used to deconvolute the data. The β7 parvalbumin isoform had a rate of synthesis that was greater than the rate of degradation. In contrast the rate of degradation of the β5 isoform exceeded its rate of synthesis, whilst the analysis revealed that the other parvalbumin β-isoforms (β1, β2, β3, β4 and β6) had a rate of synthesis that was equal to the rate of degradation. This work has addressed a number of technical challenges and represents the first study to use proteomic approaches to measure the turnover of individual proteins in fish.

Nitric oxide and l-arginine metabolism in a devascularized porcine model of acute liver failure

In acute liver failure (ALF), the hyperdynamic circulation is believed to be the result of overproduction of nitric oxide (NO) in the splanchnic circulation. However, it has been suggested that arginine concentrations (the substrate for NO) are believed to be decreased, limiting substrate availability for NO production. To characterize the metabolic fate of arginine in early-phase ALF, we systematically assessed its interorgan transport and metabolism and measured the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in a porcine model of ALF. Female adult pigs (23-30 kg) were randomized to sham (N = 8) or hepatic devascularization ALF (N = 8) procedure for 6 h. We measured plasma arginine, citrulline, ornithine levels; arginase activity, NO, and ADMA. Whole body metabolic rates and interorgan flux measurements were calculated using stable isotope-labeled amino acids. Plasma arginine decreased >85% of the basal level at t = 6 h (P < 0.001), whereas citrulline and ornithine progressively increased in ALF (P < 0.001 and P < 0.001, vs. sham respectively). No difference was found between the groups in the whole body rate of appearance of arginine or NO. However, ALF showed a significant increase in de novo arginine synthesis (P < 0.05). Interorgan data showed citrulline net intestinal production and renal consumption that was related to net renal production of arginine and ornithine. Both plasma arginase activity and plasma ADMA levels significantly increased in ALF (P < 0.001). In this model of early-phase ALF, arginine deficiency or higher ADMA levels do not limit whole body NO production. Arginine deficiency is caused by arginase-related arginine clearance in which arginine production is stimulated de novo.

Comparison of stable‐isotope labeling with amino acids in cell culture and spectral counting for relative quantification of protein expression

Protein quantification is one of the principal goals of mass spectrometry (MS)-based proteomics, and many strategies exist to achieve it. Several approaches involve the incorporation of a stable-isotope label using either chemical derivatization, enzymatically catalyzed incorporation of (18)O, or metabolic labeling in a cell or tissue culture. These techniques can be cost or time prohibitive or not amenable to the biological system of interest. Label-free techniques including those utilizing integrated ion abundance and spectral counting offer an alternative to stable-isotope-based methodologies. Herein, we present the comparison of stable-isotope labeling of amino acids in cell culture (SILAC) with spectral counting for the quantification of human embryonic stem cells as they differentiate toward the trophectoderm at three time points. Our spectral counting experimental strategy resulted in the identification of 2641 protein groups across three time points with an average sequence coverage of 30.3%, of which 1837 could be quantified with more than five spectral counts. SILAC quantification was able to identify 1369 protein groups with an average coverage of 24.7%, of which 1027 could be quantified across all time points. Within this context we further explore the capacity of each strategy for proteome coverage, variation in quantification, and the relative sensitivity of each technique to the detection of change in relative protein expression.

Primary sequence determination of a monoclonal antibody against α-synuclein using a novel mass spectrometry-based approach

A novel mass spectrometry-based workflow for de novo sequencing of a full-length monoclonal antibody (LB509) against α-synuclein is presented. This approach combines chemical modification of cysteine residues, multiple enzymatic digestion, stable isotope labeled amino acids in cell culture (SILAC), liquid chromatography coupled tandem mass spectrometry (LC/MS/MS) analysis including collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), and de novo sequencing software for data interpretation. The introduction of aminoethyl-8 (A-8) reagent which renders cysteine residues in the antibody to become substrates for trypsin digestion significantly increases the sequence coverage of the antibody, especially in the complementarity determining regions (CDRs). Incorporation of SILAC helps to distinguish minor sequence variations between original and deduced sequences. Methods in obtaining detailed sequence characterization are described, highlighting the advantage of using multiple fragmentation schemes, CID in ion trap and HCD in C-trap. Complete primary sequence for LB509 has been construed and the antibody generated exhibits specific binding to α-synuclein. The development and implementation of this technology enables an alternative approach for generating therapeutic candidate and reagent antibodies.

MicroRNA links obesity and impaired glucose metabolism

MicroRNA links obesity and impaired glucose metabolism

Dystrophin Deficiency Causes Hyper‐Secretion and Atrophy in Myotubes

The generally accepted view of the pathology of Duchenne muscular dystrophy is that absence of dystrophin renders myofibres abnormally susceptible to work-induced trauma, precipitating recurrent bouts of segmental myofibre necrosis. Necrosis stimulates inflammation and a regenerative response by myoblasts; the latter gradually becoming ineffective due to exhaustion of cells and accumulation of connective tissue driven by chronic inflammation. However, while searching for biomarkers in tissue cultures of dystrophic mouse muscle, we found phenomena that cause us to revise this picture. Using stable isotope labeled amino acids (SILAC) combined with liquid chromatography tandem mass spectrometry, we found that undamaged dystrophic myotubes are hypersecretive, excessively effluxing 57 intracellular proteins by mechanisms that cannot be identified as cell death, or generalized increase in passive leakiness. In addition dystrophic myotubes were smaller than wild-type and contain less protein and RNA per nucleus. We hypothesize that lack of dystrophin, as well as predisposing the myofibre to necrosis during physiological stress, has more profound general effects on its metabolic balance and its relationship with the extracellular environment, these potentially contributing to progressive muscle pathology. We identify myosin light chain 1 as a biomarker for in vitro drug screening. Supported by DoD.

Accurate proteome-wide protein quantification from high-resolution 15N mass spectra

In quantitative mass spectrometry-based proteomics, the metabolic incorporation of a single source of 15N-labeled nitrogen has many advantages over using stable isotope-labeled amino acids. However, the lack of a robust computational framework for analyzing the resulting spectra has impeded wide use of this approach. We have addressed this challenge by introducing a new computational methodology for analyzing 15N spectra in which quantification is integrated with identification. Application of this method to an Escherichia coli growth transition reveals significant improvement in quantification accuracy over previous methods.

Membrane-Associated RING-CH Proteins Associate with Bap31 and Target CD81 and CD44 to Lysosomes

Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins.

Trans-SILAC: sorting out the non-cell-autonomous proteome

Non-cell-autonomous proteins are incorporated into cells that form tight contacts or are invaded by bacteria, but identifying the full repertoire of transferred proteins has been a challenge. Here we introduce a quantitative proteomics approach to sort out non-cell-autonomous proteins synthesized by other cells or intracellular pathogens. Our approach combines stable-isotope labeling of amino acids in cell culture (SILAC), high-purity cell sorting and bioinformatics analysis to identify the repertoire of relevant non-cell-autonomous proteins. This 'trans-SILAC' method allowed us to discover many proteins transferred from human B to natural killer cells and to measure biosynthesis rates of Salmonella enterica proteins in infected human cells. Trans-SILAC should be a useful method to examine protein exchange between different cells of multicellular organisms or pathogen and host.

Comprehensive proteome analysis using quantitative proteomic technologies

With the completion of genome sequencing of several organisms, attention has been focused to determine the function and functional network of proteins by proteome analysis. The recent techniques of proteomics have been advanced quickly so that the high-throughput and systematic analyses of cellular proteins are enabled in combination with bioinformatics tools. Furthermore, the development of proteomic techniques helps to elucidate the functions of proteins under stress or diseased condition, resulting in the discovery of biomarkers responsible for the biological stimuli. Ultimate goal of proteomics orients toward the entire proteome of life, subcellular localization, biochemical activities, and their regulation. Comprehensive analysis strategies of proteomics can be classified as three categories: (i) protein separation by 2-dimensional gel electrophoresis (2-DE) or liquid chromatography (LC), (ii) protein identification by either Edman sequencing or mass spectrometry (MS), and (iii) quanitation of proteome. Currently MS-based proteomics turns shiftly from qualitative proteome analysis by 2-DE or 2D-LC coupled with off-line matrix assisted laser desorption ionization (MALDI) and on-line electrospray ionization (ESI) MS, respectively, to quantitative proteome analysis. Some new techniques which include top-down mass spectrometry and tandem affinity purification have emerged. The in vitro quantitative proteomic techniques include differential gel electrophoresis with fluorescence dyes, protein-labeling tagging with isotope-coded affinity tag, and peptide-labeling tagging with isobaric tags for relative and absolute quantitation. In addition, stable isotope labeled amino acid can be in vivo labeled into live culture cells through metabolic incorporation. MS-based proteomics extends to detect the phosphopeptide mapping of biologically crucial protein known as one of post-translational modification. These complementary proteomic techniques contribute to not only the understanding of basic biological function but also the application to the applied sciences for industry.

Phosphorus-Based Absolutely Quantified Standard Peptides for Quantitative Proteomics

An innovative method for the production of absolutely quantified peptide standards is described. These are named phosphorus-based absolutely quantified standard (PASTA) peptides. As the first step, synthetic phosphopeptides are calibrated via a hybrid LC-(ICP+ESI)-MS system. Quantification is achieved by ICP-MS detection of 31P, and identification is performed by ESI-MS. Generation of phosphopeptide standard solutions with this system is demonstrated to provide absolute concentrations with an accuracy better than 10%. The concept was extended to the production of peptide standards by subjecting a PASTA phosphopeptide to gentle and complete dephosphorylation to obtain the cognate PASTA peptide. It is demonstrated that both enzymatic hydrolysis by alkaline or antarctic phosphatase or chemical hydrolysis by hydrofluoric acid can be employed for this purpose. Further, the introduction of one or more stable isotope-labeled amino acids (preferably labeled by 13C, 15N) results in the production of a labeled PASTA peptide, which then can be employed as an internal standard for quantitative analysis by LC-ESI-MS. Using a 1:1 mixture of a stable isotope-labeled PASTA peptide/phosphopeptide pair as dual standard, a quantification between active and inactive recombinant MAP kinase p38alpha was performed by a combination of tryptic digestion and nanoLC-MS.

Newton's force as countermeasure for disuse atrophy

the observation that disuse leads to muscle atrophy is an old one, but we still lack a good mechanistic description of what is going on to cause wasting in conditions as diverse as bed rest and spaceflight. In fact, much of the current descriptions of the superficial mechanisms of atrophy come from

Quantitative Profiling of Polar Cationic Metabolites in Human Cerebrospinal Fluid by Reversed-Phase Nanoliquid Chromatography/Mass Spectrometry

Reversed-phase (RP) nanoliquid chromatography (LC)/mass spectrometry (MS) is widely used for proteome analysis, but hydrophilic metabolites are poorly retained on RP columns. We describe here the development and application of an efficient, robust, and quantitative nano-LC/MS method for cationic metabolome analysis in the positive ionization mode without any derivatization of analytes. Various stationary phases for nano-LC, coating of the internal wall of the capillary column, and various mobile phases were evaluated in terms of separation and peak shapes for 33 hydrophilic metabolites, including nonderivatized amino acids. Polar cationic compounds were strongly bound to mixed-functional RP with cation exchange mode resin, and the best separation was obtained with hydrophilic internal wall coating and a two-step trifluoroacetic acid (TFA) gradient in methanol as the mobile phase. Simple, but optimized, sample processing and the use of a high content of methanol allowed robust nano-LC/MS analysis. Our developed method was applied for biomarker discovery in Alzheimer's disease (AD). Several hundred peaks were detected from 10 microL of cerebrospinal fluid (CSF). In a principal component analysis (PCA) plot using peak intensities without normalization, peak separation depended on the experimental date, not disease state. Therefore, constant amounts of two stable isotope-labeled amino acids, Val and Lys, were added as internal standards (ISs) to each sample before processing. These ISs were eluted in different gradient slopes in the two-step gradient, and the normalized peak ratios using the corresponding ISs gave a unique group of PCA scores which could distinguish AD CSF samples from age-matched control CSF samples.

Turnover of the Human Proteome: Determination of Protein Intracellular Stability by Dynamic SILAC

The proteome of any system is a dynamic entity, such that the intracellular concentration of a protein is dictated by the relative rates of synthesis and degradation. In this work, we have analyzed time-dependent changes in the incorporation of a stable amino acid resolved precursor, a protocol we refer to as "dynamic SILAC", using 1-D gel separation followed by in-gel digestion and LC-MS/MS analyses to profile the intracellular stability of almost 600 proteins from human A549 adenocarcinoma cells, requiring multiple measures of the extent of labeling with stable isotope labeled amino acids in a classic label-chase experiment. As turnover rates are acquired, a profile can be built up that allows exploration of the 'dynamic proteome' and of putative features that predispose a protein to a high or a low rate of turnover. Moreover, measurement of the turnover rate of individual components of supramolecular complexes provides a unique insight in processes of protein complex assembly and turnover.