Complex Mixture Of Amino Acids Research Articles

Heat flows enrich prebiotic building blocks and enhance their reactivity

The emergence of biopolymer building blocks is a crucial step during the origins of life1–6. However, all known formation pathways rely on rare pure feedstocks and demand successive purification and mixing steps to suppress unwanted side reactions and enable high product yields. Here we show that heat flows through thin, crack-like geo-compartments could have provided a widely available yet selective mechanism that separates more than 50 prebiotically relevant building blocks from complex mixtures of amino acids, nucleobases, nucleotides, polyphosphates and 2-aminoazoles. Using measured thermophoretic properties7,8, we numerically model and experimentally prove the advantageous effect of geological networks of interconnected cracks9,10 that purify the previously mixed compounds, boosting their concentration ratios by up to three orders of magnitude. The importance for prebiotic chemistry is shown by the dimerization of glycine11,12, in which the selective purification of trimetaphosphate (TMP)13,14 increased reaction yields by five orders of magnitude. The observed effect is robust under various crack sizes, pH values, solvents and temperatures. Our results demonstrate how geologically driven non-equilibria could have explored highly parallelized reaction conditions to foster prebiotic chemistry.

Fully united, easy, and economical sensor array for newborn babies’ amino acids monitoring: Identification of amino acids in healthy and unhealthy with PKU newborn babies

Amino acid (AA) disorders are the main class of inborn errors of metabolism with diverse medical presentations. This paper was aimed to provide a novel and efficient sensor array for the quantification and differentiation of AAs using different pH buffer solutions as sensor elements (SEs) and nanocurcumin (NC) in the role of a marker in biofluids of newborn babies. Amino and carboxyl groups along with the side chain of different AAs in different pH buffer solutions are protonated or deprotonated. This makes each AA molecule an acid (in the role of a proton donor), a hydrogen bonding donor, as well as a polar ion. So that, these differences may permit a profile differentiation-based sensor array for AAs discrimination. Using NC as a marker, the interactions between AA and NC in different pH buffer solutions (mainly involved in acid-base, hydrogen bonding, and π- π stacking interactions) result in absorbance changes, making a discriminate response profile for each AA. The results reveal that AAs can be discriminated successfully at three concentrations levels 10, 25, and 50 µM by linear discrimination analysis (LDA) and hierarchical clustering analysis (HCA). Complex AA mixtures are also capable to be classified. The results show that our sensor array can be potentially employed to the differentiation of AAs in biofluids of healthy and unhealthy newborn babies. It should be noted that the sensor array requires only common and available lab equipment and materials, which can be applied in AAs-related fundamental studies and clinical diagnosis.

Why is the Sensory Response of Organic Probes within a Polymer Film Different in Solution and in the Solid-State? Evidence and Application to the Detection of Amino Acids in Human Chronic Wounds.

We anchored a colourimetric probe, comprising a complex containing copper (Cu(II)) and a dye, to a polymer matrix obtaining film-shaped chemosensors with induced selectivity toward glycine. This sensory material is exploited in the selectivity detection of glycine in complex mixtures of amino acids mimicking elastin, collagen and epidermis, and also in following the protease activity in a beefsteak and chronic human wounds. We use the term inducing because the probe in solution is not selective toward any amino acid and we get selectivity toward glycine using the solid-state. Overall, we found that the chemical behaviour of a chemical probe can be entirely changed by changing its chemical environment. Regarding its behaviour in solution, this change has been achieved by isolating the probe by anchoring the motifs in a polymer matrix, in an amorphous state, avoiding the interaction of one sensory motif with another. Moreover, this selectivity change can be further tuned because of the effectiveness of the transport of targets both by the physical nature of the interface of the polymer matrix/solution, where the target chemicals are dissolved, for instance, and inside the matrix where the recognition takes place. The interest in chronic human wounds is related to the fact that our methods are rapid and inexpensive, and also considering that the protease activity can correlate with the evolution of chronic wounds.

Enantioselective multiple heartcut two-dimensional ultra-high-performance liquid chromatography method with a Coreshell chiral stationary phase in the second dimension for analysis of all proteinogenic amino acids in a single run

A multiple heartcut (MHC) 2D-UHPLC method with UV detection has been developed for the enantioselective analysis of complex amino acid mixtures in a single run. The MHC method is based on an achiral gradient RPLC separation with 1.8 μm C18 phase (100 × 2.1 mm ID column) in the first dimension (1D) and enantioselective isocratic separation on a tert-butylcarbamoylquinine-based 2.7 μm Coreshell particle column (50 × 3 mm ID) in the second dimension (2D). Pre-column derivatization has been performed with Sanger’s reagent (2,4-dinitrofluorobenzene) yielding chromogenic 2,4-dinitrophenylated amino acids (DNP-AAs). Heartcuts of 40 μL fractions of the 1D peaks were sampled into the 2D system via a two-position four-port dual valve connected to two loop decks each equipped with six 40 μL parking loops. Using this setup, 25 amino acids (20 proteinogenic plus allo-Thr, allo-Ile, homoserine (Hse), Orn, β-Ala) have been analyzed enantioselectively in a fully automated manner with a single chiral column within 130 min total run time (1D and 2D). All 2D separations together took 101.5 min (29 cuts with 3.5 min run time each) and thus the total analysis time was quite efficiently utilized. Faster separations were restricted by some software constraints which did not allow to adjust run times in 2D individually. The practical utility of this enantioselective MHC method is documented by application for the absolute configuration determination of the amino acids in gramicidin and bacitracin. Further optimizations should lead to a generic enantioselective amino acid analyzer for the quality control of synthetic peptides and the structural characterization of non-ribosomal peptides.

Simultaneous determination of natural‐abundance δ15N values and quantities of individual amino acids in proteins from milk of lactating women and from infant hair using gas chromatography/isotope ratio mass spectrometry

In isotope tracer experiments used in nutritional studies, it is frequently desirable both to determine the (15)N/(14)N ratios of target compounds and to quantify these compounds. This report shows how this can be achieved in a single chromatographic run for protein amino acids using an isotope ratio mass spectrometer. Protein hydrolysis by acidic digestion was used to release amino acids, which were then derivatized as their N-pivaloyl-O-isopropyl esters. Suitable conditions for sample preparation were established for both hair and milk proteins. The N-pivaloyl-O-isopropyl esters of amino acids were separated by gas chromatography (GC) on a 60 m ZB-WAX column linked via a combustion interface to an isotope ratio mass spectrometer. The (15)N/(14)N ratios were obtained from the m/z 28, 29 and 30 peak intensities and the quantities from the Area All (Vs) integrated peak areas. It is shown from a five-point calibration curve that both parameters can be measured reliably within the range of 1.0 to 2.0 mg/mL for the major amino acids derived from the hydrolysis of human maternal milk or hair samples. The method was validated in terms of inter-day and inter-user repeatability for both parameters for 14 amino acids. The amino acid percentage composition showed a good correlation with literature values. The method was applied to determine the variability in a population of lactating mothers and their infants. It has been established that δ(15)N values can be simultaneously determined for a complex mixture of amino acids at variable concentrations. It is shown that the percentage composition obtained correlates well with that obtained by calculation from the protein composition or from literature values. This procedure should provide a significant saving in analysis time, especially in those cases where the GC run-time is necessarily long. It allows the satisfactory determination of the variation within a sample population.

Separation of complex mixtures of amino acids for biorefinery applications using electrodialysis

The potential of electrodialysis (ED) for the separation of amino acids (zwitterionic molecules that exhibit a specific charge behavior dependent on pH) has been demonstrated in the past. However, even though successful for the separation of specific amino acids, ED is not applicable for the separation of mixtures containing the positively charged amino acid arginine (Arg) due to its poisonous effect on commercially available cation exchange membranes (CEMs), while Arg is one of the major components in biobased feeds and an important precursor for the production of chemicals. The present study confirms that this inhibiting effect is directly related to the water content of the cation exchange membrane during the ED experiments. To overcome this limitation, ED with self-prepared cation exchange membranes with a high swelling degree (SPEEK) and ED with ultrafiltration membranes (EDUF) was applied. The results clearly prove that the separation of lysine (Lys) and Arg is possible with the two proposed alternative ED configurations. Separation of more complex biobased amino acid mixtures containing Arg showed the simultaneous separation of the acidic amino acids, glutamic acid (Glu) and aspartic acid (Asp), and the basic amino acids, Lys and Arg, from neutral alanine (Ala) validating the potential of ED for biorefinery applications.

Separation of L-aspartic acid and L-glutamic acid mixtures for use in the production of bio-based chemicals

BACKGROUND: Amino acids are promising feedstocks for the chemical industry due to their chemical functionality. They can be obtained by the hydrolysis of potentially inexpensive protein streams such as the byproduct of biofuel production. However, individual amino acids are required before they can be used for the further production of chemicals. Here, the separation of L-aspartic acid (Asp) and L-glutamic acid (Glu) mixture, which can be isolated from protein hydrolysis solutions at low pH or from electrodialysis of complex amino acid mixtures, was studied. RESULTS: Glu was converted into L-pyroglutamic acid (pGlu) which can be separated from the mixture of Asp and Glu due to its higher solubility in water. The conversion was carried out under aqueous or melt conditions. Under aqueous conditions, the conversion was studied as a factor of time, temperature and the amount of Glu. The conversion was specific with high yield and not effected by Asp. After pGlu was separated from Asp and residual Glu by solubility difference, it can be transferred back to Glu through hydrolysis. CONCLUSION: The conversion of Glu to pGlu is specific and can be applied to separation Asp and Glu for their use in the production of bio-based chemicals

Olfactory discrimination of complex mixtures of amino acids by the black bullhead Ameiurus melas

On the basis of previous findings of behavioural discrimination of amino acids and on the knowledge of electrophysiology of the catfish (genera Ictalurus and Ameiurus) olfactory organs, behavioural experiments that investigated olfactory discrimination of amino acid mixtures were carried out on the black bullhead Ameiurus melas. Repeated presentations of food-rewarded mixtures released increased swimming activity measured by counting the number of turns >90° within 90 s of stimulus addition. Non-rewarded amino acids and their mixtures released little swimming activity, indicating that A. melas discriminated between the conditioned and the non-conditioned stimuli. Two questions of mixture discrimination were addressed: (1) Are A. melas able to detect components within simple and complex amino acid mixtures? (2) What are the smallest differences between two complex mixtures that A. melas can detect? Three and 13 component mixtures tested were composed primarily of equipotent amino acids [determined by equal electroolfactogram (EOG) amplitude] that contained L-Cys at ×100 the equipotent concentration. Ameiurus melas initially perceived the ternary amino acid mixture as its more stimulatory component alone [i.e. cysteine (Cys)], whereas the conditioned 13 component mixture containing the more stimulatory L-Cys was perceived immediately as different from L-Cys alone. The results indicate that components of ternary mixtures are detectable by A. melas but not those of more complex mixtures. To test for the smallest detectable differences in composition between similar multimixtures, all mixture components were equipotent. Initially, A. melas were unable to discriminate the mixtures of six amino acids from the conditioned mixtures of seven amino acids, whereas they discriminated immediately the mixtures of four and five amino acids from the conditioned mixture. Experience with dissimilar mixtures enabled the A. melas to start discriminating the seven-component conditioned mixture from its six-component counterparts. After fewer than five training trials, A. melas discriminated the mixtures of nine and 10 amino acids from a conditioned mixture of 12 equipotent amino acids; however, irrespective of the number of training trials, A. melas were unable to discriminate the 12 component mixture from its 11 component counterparts.

Transport activity–dependent intracellular sorting of the yeast general amino acid permease

Intracellular trafficking of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae is regulated by amino acid abundance. When amino acids are scarce Gap1p is sorted to the plasma membrane, whereas when amino acids are abundant Gap1p is sorted from the trans-Golgi through the multivesicular endosome (MVE) and to the vacuole. Here we test the hypothesis that Gap1p itself is the sensor of amino acid abundance by examining the trafficking of Gap1p mutants with altered substrate specificity and transport activity. We show that trafficking of mutant Gap1p(A297V), which does not transport basic amino acids, is also not regulated by these amino acids. Furthermore, we have identified a catalytically inactive mutant that does not respond to complex amino acid mixtures and constitutively sorts Gap1p to the plasma membrane. Previously we showed that amino acids govern the propensity of Gap1p to recycle from the MVE to the plasma membrane. Here we propose that in the presence of substrate the steady-state conformation of Gap1p shifts to a state that is unable to be recycled from the MVE. These results indicate a parsimonious regulatory mechanism by which Gap1p senses its transport substrates to set an appropriate level of transporter activity at the cell surface.

Cellular Binding of Nanoparticles in the Presence of Serum Proteins

Nanoparticles have important biomedical applications ranging from the treatment of human disease with gene therapy to understanding basic cellular function. For cellular uptake of nanoparticles to occur, the particle must first bind to the cell surface. We sought to understand how nanoparticles interact with the cell surface in the presence of serum proteins. Cell culture medium supplemented with serum is a highly complex mixture of amino acids, vitamins, inorganic salts, glucose and proteins. Characterization of cationic, fluorescent nanoparticles in the presence of cell culture medium demonstrated that the particles rapidly bind a mixture of proteins present in the serum resulting in an anionic nanoparticle that essentially presents a surface of serum proteins to the cell surface. Probing the interaction of this serum protein-nanoparticle complex with the cell surface was achieved using fluorescently labeled proteins and monitoring the colocalization of nanoparticle and protein signal with two-color fluorescence microscopy over time. Our results indicated that both nanoparticle and protein were highly colocalized at early times and this was found to decrease with increased incubation time. Displacement of the serum proteins from the nanoparticles was found to be protein dependent. This study has important implications for the rational design of nanoparticles and ligands for delivery in a cellular environment as it demonstrates the significance of serum protein-nanoparticle interactions. Such interactions can ultimately influence the cellular binding of the nanoparticle to the cell surface.[1][1] G. W. Doorley and C. K. Payne, “ Cellular Binding of Nanoparticles in the Presence of Serum Proteins” Chem. Commun., 2010, in press

Enantiomeric separation of ornithine in complex mixtures of amino acids by EKC with off-line derivatization with 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate

A new analytical methodology was developed by EKC enabling the fast enantiomeric separation of Ornithine in complex mixtures of amino acids. A previous derivatization step with 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate (AQC) was achieved to enable the sensitive UV detection of amino acids as well as to make possible their interaction with the CDs employed as chiral selectors. A dual CD system containing an anionic and a neutral CD in phosphate buffer at acid pH showed a high resolving power allowing the enantiomeric separation of 18 protein amino acids and Orn. The method was applied to the analysis of fermented foods to investigate the extent of the presence of Orn enantiomers.

Chiral high-performance liquid chromatography analysis of α-amino acid mixtures using a novel SH reagent— N- R-mandelyl- l-cysteine and traditional enantiomeric thiols for precolumn derivatization

Several chiral thiols, i.e. traditionally used enantiomerically pure SH reagents and novel N- R-mandelyl- l-cysteine ( R-NMC) containing additional chiral center, have been applied as co-reagents in precolumn derivatization with o-phthalaldehyde for enantiomeric HPLC analysis of individual α-amino acids and their mixtures. The R-NMC-derived isoindoles as well as adducts with other thiols have a characteristic absorption maximum at 340 nm, and are highly fluorescent allowing detection of 10 μg/l of an amino acid. Investigated 19 amino acids were analyzed separately and in a mixture by a gradient HPLC after precolumn derivatization. The chromatographic behavior of formed isoindoles substantially differs for each of the thiols used for modification. In contrast to traditional enantiomeric thiols application of diastereomeric R-NMC provides higher resolution for α-amino acid enantiomers, with l, d-elution order (except for Arg). Combined use of R-NMC and other thiol enlarges the possibilities of this method, allowing accurate chiral analysis of complex amino acid mixtures.

Simultaneous Screening of Multiple Bacterial tRNA Synthetases Using an Escherichia coli S30-Based Transcription and Translation Assay

The search for novel antibiotics to combat the growing threat of resistance has led researchers to screen libraries with coupled transcription and translation systems. In these systems, a bacterial cell lysate supplies the proteins necessary for transcription and translation, a plasmid encoding a reporter protein is added as a template, and a complex mixture of amino acids and cofactors is added to supply building blocks and energy to the assay. Firefly luciferase is typically used as the reporter protein in high-throughput screens because the luminescent signal is strong and, since bacterial lysates contain no luciferase, the background is negligible. The typical coupled transcription and translation assay is sensitive to inhibitors of RNA polymerase and to compounds that bind tightly to the ribosome. We have found a way to increase the information content of the screen by making the assay more sensitive to inhibitors of tRNA synthetases. Restricting the concentration of amino acids added to the reaction mixture allows the simultaneous screening of multiple tRNA synthetase enzymes along with the classic transcription and translation targets. In addition, this assay can be used as a convenient way to determine if an antibacterial compound of unknown mechanism inhibits translation through inhibition of a tRNA synthetase, and to identify which synthetase is the target.

Direct analysis of 15N‐label in amino and amide groups of glutamine and asparagine

A novel method for on-line determination of the amount and position of 15N-labeling in complex mixtures of amino acids is presented. Underivatized amino acids were analyzed by ion-pair chromatography in combination with mass spectrometry. This enables the direct determination of the 15N label distribution. The fragmentation pathways of the nitrogen moieties of glutamine (Gln) and asparagine (Asn) were studied in detail using all mono 15N isotopomers, which led to a method for differentiating between 15N-amide and 15N-amino labeling. The fragmentation involving the amino and amide groups of Gln led to distinct ion structures. The equivalent fragmentation pattern was not observed for Asn. Instead, the amide group of Asn was eliminated as HNCO in a secondary process. The developed analytical method was evaluated by analysis of a range of standard mixtures taking into account different levels of 15N abundance and distribution between the amino and amide groups. The detection limit (3 SD) for the presence of a 15N label was 0.7 and 1.0% for Gln and Asn, respectively. The determination of the positional labeling follows a nonlinear function. A representative example at 30% 15N was used as a benchmark resulting in average relative standard deviations of 2.7 and 15% for Gln and Asn, respectively. The corresponding expectation windows for the positional labeling were found to be 2 and 12%, respectively.

Functional Analysis of the Carboxy-Terminal Region of Bacillus subtilis TnrA, a MerR Family Protein

The Bacillus subtilis TnrA transcription factor belongs to the MerR family of proteins and regulates gene expression during nitrogen-limited growth. When B. subtilis cells are grown with excess nitrogen, feedback-inhibited glutamine synthetase forms a protein-protein complex with TnrA that prevents TnrA from binding to DNA. The C-terminal region of TnrA is required for the interaction with glutamine synthetase. Alanine scanning mutagenesis of the C-terminal region of TnrA identified three classes of mutants that altered the regulation by glutamine synthetase. While expression of the TnrA-regulated amtB gene was expressed constitutively in the class I (M96A, Q100A, and A103G) and class II (L97A, L101A, and F105A) mutants, the class II mutants were unable to grow on minimal medium unless a complex mixture of amino acids was present. The class III tnrA mutants (R93A, G99A, N102A, H104A, and Y107A mutants) were partially defective in the regulation of TnrA activity. In vitro experiments showed that feedback-inhibited glutamine synthetase had a significantly reduced ability to inhibit the DNA-binding activity of several class I and class II mutant TnrA proteins. A coiled-coil homology model of the C-terminal region of TnrA is used to explain the properties of the class I and II mutant proteins. The C-terminal region of TnrA corresponds to a dimerization domain in other MerR family proteins. Surprisingly, gel filtration and cross-linking analysis showed that a truncated TnrA protein which contained only the N-terminal DNA binding domain was dimeric. The implications of these results for the structure of TnrA are discussed.

In Vitro Selection of Membrane-spanning Leucine Zipper Protein-Protein Interaction Motifs Using POSSYCCAT

A membrane-spanning heptad repeat motif mediates interaction between transmembrane segments. This motif was randomized with three different sets of mostly hydrophobic residues in the context of POSSYCCAT, a modified ToxR transcription activator system. The resulting combinatorial libraries were subjected to different levels of selective pressure to obtain groups of transmembrane segments that are distinguished by their ability to self-interact in bacterial membranes. Upon relating self-interaction to amino acid composition, the following conclusions were made. First, randomization with only Leu, Ile, Val, Met, and Phe resulted in unexpected robust self-interaction with little sequence specificity. Second, with more complex amino acid mixtures that represent natural transmembrane segments more closely, self-interaction critically depended on amino acid composition of the interface. Whereas the contents of Ile and Leu residues increased with the ability to self-interact, the contents of Pro and Arg residues decreased. Third, heptad repeat motifs composed of Leu, Ile, Val, Met, and Phe were approximately 40-fold over-represented in transmembrane segments of single-span membrane proteins as compared with motifs composed of the more complex amino acid mixtures. This suggests that heptad motifs composed of the smaller subset of amino acids were enriched in the course of natural single-span membrane protein evolution.

Electrophysiological responses of taste cells to nutrient mixtures in the polyphagous caterpillar of Grammia geneura.

In a normally feeding insect, the taste receptors are exposed to complex mixtures of chemicals, not single compounds. We investigate the responses of neurons in the galeal sensilla of the caterpillar of Grammia geneura to mixtures of nutrient compounds at concentrations occurring in plants. Compounds that stimulated the same neuron were generally additive in their effects in binary mixtures. Amino acids that did not stimulate usually had no effect in mixtures with a stimulating compound, but glutamic acid reduced the response to serine in the medial sensillum. Nutrient compounds that stimulated different cells in a sensillum acted independently of each other. Complex mixtures of amino acids resembling samples of free amino acids from three host plants were less stimulating than expected from their molar concentrations. In host plant selection, the response from the medial sensillum is probably dominated by sucrose; unless sucrose levels are low, amino acids will contribute little to sensory input because they stimulate the same cell as sucrose. In the lateral sensillum, amino acids act independently of sugars. The limited contact chemosensory array of caterpillars seems inadequate to allow them to make fine distinctions between plants on the basis of their free amino acids.

Nutrients, Signals, and Photosynthate Release by Symbiotic Algae (The Impact of Taurine on the Dinoflagellate Alga Symbiodinium from the Sea Anemone Aiptasia pulchella)

Exogenous concentrations of 10 [mu]M to 1 mM of the nonprotein amino acid taurine stimulated photosynthate release from the dinoflagellate alga Symbiodinium, which had been freshly isolated from the sea anemone Aiptasia pulchella. Photosynthate release, as induced by taurine and animal extract, was metabolically equivalent at both concentrations in that they (a) stimulated photosynthate release to the same extent and (b) induced the selective release of photosynthetically derived organic acids. A complex mixture of amino acids at 75 mM also promoted photosynthate release, but the release rate was reduced by 34% after the omission of taurine (3 mM) from the mixture, suggesting that much of the effect of amino acids was largely attributable to taurine. Exogenous 14C-labeled taurine was taken up by the cells, and more than 95% of the internalized 14C was recovered as taurine, indicating that taurine-induced photosynthate release was not dependent on taurine metabolism. Both taurine uptake and taurine-induced photosynthate release by Symbiodinium exhibited saturation kinetics, but with significantly different Km values of 68 and 21 [mu]M, respectively. The difference in Km values is compatible with the hypothesis that Symbiodinium has a taurine signal transducer that is responsible for photosynthate release and is distinct from the taurine transporter.

Amino acids interfere with the ninhydrin assay for asparagine

A colorimetric ninhydrin assay, reportedly specific for asparagine, has been published (Sheng et al. (1993). Anal. Biochem., 211, 242–924). The suitability of this assay for determining asparagine in food samples was tested by using it to measure the asparagine levels in tips of harvested asparagus spears. Compared with our standard chromatographic procedure ( x), the colorimetric assay ( y) severely overestimated asparagine in asparagus ( y = 129·6 + 1·12 x, S y x = 32·2 ) even though the results from the two methods were highly correlated ( r = 0·997, P = 0·003). By testing other amino acids in the assay, we showed that they also react with ninhydrin under the conditions of Sheng et al. In particular, proline gave a response two-fold greater than asparagine and the response with phenylalanine was half that of asparagine. It is believed that the overestimation of asparagine by the ninhydrin method is due to the additive response of the other amino acids present in asparagus. It is concluded that the assay is unsuitable for the routine determination of asparagine in asparagus, or in complex mixtures of amino acids in other foods.

2H- and13C-labeled amino acids generated by obligate methylotrophs Biosynthesis and MS monitoring

Biosynthetic preparation of(2)H- and(13)C- labeled amino acids was studied using a leucine-producing mutant of the obligate methylotroph,Methylobacillus flagellatum. The strain was cultivated in various media containing(13)C- or(2)H-analogs of methanol. The total protein from each experiment was subjected to acid hydrolysis and converted into a mixture of dansyl amino acid methyl esters. The samples of excreted leucine were converted into methyl esters of dansyl and benzyloxycarbonyl derivatives. Electron impact mass spectrometry was performed to detect stable isotope enrichment of the amino acids. According to the mass spectrometric analysis it is feasible to use methylotrophic microorganisms for the preparation of(2)H- and(13)C- analogs of amino acids by labeled methanol bioconversion; the excreted amino acids can be convenient for express analysis as an indicator of isotopic enrichment of the total protein. The data obtained testified to a high efficiency of dansyl derivatization for mass spectrometric analysis of complex amino acid mixtures.