Search for amino acids in apollo returned lunar soil

The lunar samples from Apollo flights 11 through 17 provided the students of chemical evolution with an opportunity of examining extraterrestrial materials for evidence of early prebiological chemistry in the solar system. Our search was directed to water-extractable compounds with emphasis on amino acids. Gas chromatography, ion-exchange chromatography and gas chromatography combined with mass spectrometry were used for the analysis. It is our conclusion that amino acids are not present in the lunar regolith above the background levels of our investigations.     

Problems in the search for amino acids in lunar fines

In the search for amino acids in lunar fines, a major problem is the prevention of contamination from terrestrial sources, and the recognition of terrestrial contamination when it has occurred. Synthesis of amino acids conceivably could take place in the lunar module rocket exhaust, a possibility that has not been adequately ruled out. Amino acids could be shed from the astronauts suits, a possibility which has not been studied at all. Amino acids could also be introduced, at many stages of terrestrial manipulation, and during the analytical procedures employed. Hand contamination has qualitative and quantitative features that are characteristic and can be assessed. Precautions for elimination of hand and microbial contamination from glassware, reagents and water are proposed.A second major problem is the efficiency of recovery of amino acids added to lunar material, which is then subject to the complete analytical scheme. This necessitates the availability of lunar material to develop proper procedures.Besides the amino acids present in excess of blank values, it is necessary for the correct interpretation of any positive findings to know whether the amino acids are free or bound and optically inactive or active.The ion exchange chromatography and gas chromatography-mass spectrometry are procedures that complement each other. Both should be applied not only to the same sample but to the same preparations. To pit one method against the other is to risk losing the best analytical data.     

On the abiotic formation of amino acids I. HCN as a precursor of amino acids detected in extracts of lunar samples II. Formation of HCN and amino acids from simulated mixtures of gases released from lunar samples

Two studies on the abiotic formation of amino acids are presented. The first study demonstrates the role of hydrogen cyanide as a precursor of amino acids detected in extracts of lunar samples. The formation of several amino acids, including glycine, alanine, aspartic acid, and glutamic acid, under conditions similar to those used for the analysis of lunar samples is demonstrated. The second study investigates the formation of hydrogen cyanide as well as amino acids from lunar-sample gas mixtures under electrical discharge conditions. These results extend the possibility of synthesis of amino acids to planetary bodies with primordial atmospheres less reducing than a mixture of methane, ammonia, hydrogen and water.     

Research for amino acids in lunar samples

Water extracts of lunar fines were analyzed for amino acids by a gas-liquid chromatographic technique whereby amino acids were converted to the N-trifluoroacetyln-butyl, esters prior to analysis. The lunar material studied included both Apollo 14 (14240 SESC and 14298) and Apollo 12 (12023) samples. The water extract of the special Apollo 14 sample (14240 SESC) was analyzed both for free and bound amino acids (hydrolysis with 6 N hydrochloric acid). In both the hydrolyzed and unhydrolyzed extracts, the amino acids were not observed above background levels.The analysis of Apollo 12 and 14 samples (12023 14298) yielded similar results. Detection limits were established at 300 pg to 1 ng for different amino acids. A large chromatographic peak with a retention temperature of 126°C was observed on analysis of sample, (12023); it was identified as oxalic acid by GC-MS. The concentration of amino acids in the Apollo 14 SESC samples processed and analyzed in the joint experiments at Ames by GLC and IEC were found to be extremely low (glycine at 3 to 4 ng g^–1). As the quantities were so minute, these identifications could not be confirmed by GLC-MS and therefore should still be considered as tentative. Other studies included the analysis of performance standards at the 2 to 6 ng level of each of 17 amino acids, and the analysis of 5 ml of H₂O containing 2 ppb of each amino acid. Recovery of amino acids added to lunar fines were conducted at the 10, 50, and 70 ng level of each amino acid with 50 to 70 mg of lunar material. The recoveries varied from as high as 80% for some of the aliphatics to complete loss of the amino acids ornithine and lysine.Contributed from Missouri Agricultural Experiment Station Journal Series No. 6255. Approved by the Director. Supported in part by grants from the National Aeronautics and Space Administration (NGR 26-004-011) and the Experiment Station Chemical Laboratories.     

Review of methods used in lunar organic analysis: Extraction and hydrolysis techniques

Extraction, hydrolysis and crushing procedures have proven useful in analyzing for some of the carbon-containing compounds which are present in Apollo 11 and 12 lunar samples. Three main extraction methods employed with aqueous and nonaqueous solvents were refluxing in open and closed systems, Soxhlet extraction, and sonication. With water and acids, refluxing was the method of choice. Of the various nonaqueous solvent systems used, benzene: methanol mixtures were most often selected, and sonication was favored over Soxhlet extraction. Extraction of lunar samples with water followed by acid hydrolysis of the water extract was found to be superior to direct acid hydrolysis of lunar material in the search for amino acids or their precursors. Direct acid hydrolysis of lunar materials did demonstrate however, that carbides or carbide-like compounds are present on the moon. Hydrolysis with deuterated acids and bases showed that lunar samples contain indigenous methane and ethane and confirmed the presence of carbide-like materials. Crushing experiments also showed that gaseous hydrocarbons can be released from lunar samples.     

Amino acids attached to transfer ribonucleic acid in vivo.

1. tRNA was extracted from rabbit liver by both the phenol and diethyl pyrocarbonate methods under conditions preventing deacylation of the amino acids attached in vivo. 2. After deacylation 12 amino acids were determined by gas-liquid chromatography, by using the flame-ionization and nitrogen-sensitive thermionic detectors. 3. Comparison of the distribution of 12 amino acids attached to tRNA with those contained in total tissue protein and in the free pool showed little correlation. 4. Results for the enzymic charging assay for tRNA in vitro did not correlate satisfactorily with the analysis of amino acids attached to tRNA in vivo. Marked differences were ntoed in comparison made between our own and other published results.     

Ion-exchange chromatography in lunar organic analysis

Although ion exchange chromatography has been used in separating amino acids from mineral salts, quantitative recovery has not been possible for the basic amino acids or for subnanomole concentrations of amino acids.As an analytical tool for amino acid analysis, ion-exchange chromatography has made it possible to resolve a relatively complex mixture of amino acids in less than an hour with detection limits of less than 10^–12 moles of amino acids. Reasonable specificity for amino acids is achieved by multiple wavelength detection of the reaction product found with ninhydrin. Unequivocal specificity must be obtained in conjunction with other methods such as mass spectrometry.In the analysis of subnanomole levels of amino acids, it is necessary to carry both reagent blanks and low-level amino acid standards through the entire sample preparation step since both contamination and selective losses occur and must be monitored.     

Pollen feeding in the butterfly Heliconius charitonia: isotopic evidence for essential amino acid transfer from pollen to eggs

Heliconius and Laparus butterflies exhibit a unique pollen-collecting behaviour that enhances lifespan and fecundity. The specific nutritional contribution of pollen, however, had not been previously demonstrated. We used stable isotope variation to trace the carbon flow into eggs from corn pollen provided experimentally to ovipositing female Heliconius charitonia, and to evaluate the use of isotopically contrasting nectar sugars in egg amino acids. The delta(13)C of individual amino acids from pollen, larval host plant and the eggs from experimental butterflies was measured with gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), to evaluate amino acid transfer. The delta(13)C of egg essential amino acids indicated a transfer of essential amino acids from pollen to butterfly eggs. However, the delta(13)C of non-essential amino acids reflected the isotopic composition of the artificial nectar, indicating that H. charitonia synthesizes non-essential amino acids from dietary sugars. This, to our knowledge, is the first direct demonstration of amino acid transfer from pollen to butterfly eggs, and suggests that essential amino acids in particular are a key resource for extending lifespan and fecundity in Heliconius butterflies.     

Chiral separation of amino acids in biological fluids by micellar electrokinetic chromatography with laser-induced fluorescence detection

A method is presented for the chiral analysis of amino acids in biological fluids using micellar electrokinetic chromatography (MEKC) and laser-induced fluorescence (LIF). The amino acids are derivatized with the chiral reagent (+/−)-1-(9-anthryl)-2-propyl chloroformate (APOC) and separated using a mixed micellar separation system. No tedious pre-purification of samples is required. The excellent separation efficiency and good detection capabilities of the MEKC-LIF system are exemplified in the analysis of urine and cerebrospinal fluid. This is the first time MEKC has been reported for chiral analysis of amino acids in biological fluids. The amino acids -alanine, -glutamine, and -aspartic acid have been observed in cerebrospinal fluid, and -alanine and -glutamic acid in urine. To the best of our knowledge no measurements of either -alanine in cerebrospinal fluid or -glutamic acid in urine have been presented in the literature before.     

High-performance liquid chromatography of side-chain-protected amino acid phenylthiohydantoins

Eighteen side-chain-protected amino acids, routinely employed in solid-phase peptide synthesis, were derivatized to their phenylthiohydantoins (PTH) by one cycle of the Edman degradation. All of these side-chain-protected PTH amino acids elute, with almost-baseline resolution, in less than 18 min by high-performance liquid chromatography, utilizing a biphasic gradient of acetonitrile in 0.01 n sodium acetate, pH 4.5, or a linear gradient of 0 to 100% acetonitrile with the exception of the coelution of a O-benzyl-threonine and carbobenzoxy-lysine phenylthiohydantoin amino acids. The derivatized amino acids were subjected to reverse-phase chromatography on a Zorbax ODS column and monitored at 254 nm. None of the PTH amino acids coelute with side-chain-protected PTH amino acid counterparts, although PTH-tosyl-histidine undergoes deprotection to PTH-histidine in the Edman degradation. A protected decapeptide attached to a chloromethylated polystyrene resin was degraded on a solid-phase sequencer in 16 h. The PTH amino acids resulting from the automated Edman degradation on the decapeptide were fully resolved and quantified in less than 3 h demonstrating that automated high-performance liquid chromatography can keep pace with both the automated sequencer and synthesizer which requires minimally 2–3 h for attachment of each residue to the growing peptide chain.     

Gas chromatographic determination of free amino acids and amino acids attached to transfer RNA in biological samples

A method was developed to analyze quantitatively free amino acids and amino acids attached to transfer RNA (tRNA) in tissue samples by gas chromatography. Free amino acids were purified by ion-exchange chromatography after deproteinization. Total cellular aminoacyl-tRNA was extracted from rabbit reticulocytes and liver by a modified phenol extraction method under conditions which were designed to prevent deacylation of the attached amino acids. After deacylation and separation from tRNA by pressure ultrafiltration, eighteen amino acids were determined by gas chromatography as their N-heptafluorobutyryl isobutyl derivatives.     

Quantitative urine amino acid analysis using liquid chromatography tandem mass spectrometry and aTRAQ reagents

Ion-exchange chromatography (IEC) is the most widely used method for amino acid analysis in physiological fluids because it provides excellent separation and reproducibility, with minimal sample preparation. The disadvantage, however, is the long analysis time needed to chromatographically resolve all the amino acids. To overcome this limitation, we evaluated a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method, which utilizes aTRAQ reagents, for amino acid analysis in urine. aTRAQ reagents tag the primary and secondary amino groups of amino acids. Internal standards for each amino acid are also labeled with a modified aTRAQ tag and are used for quantification. Separation and identification of the amino acids is achieved by liquid chromatography tandem mass spectrometry using retention times and mass transitions, unique to each amino acid, as identifiers. The run time, injection-to-injection, is 25 min, with all amino acids eluting within the first 12 min. This method has a limit of quantification (LOQ) of 1 μmol/L, and is linear up to 1000 μmol/L for most amino acids. The Coefficient of Variation (CV) was less than 20% for all amino acids throughout the linear range. Method comparison demonstrated concordance between IEC and LC-MS/MS and clinical performance was assessed by analysis of samples from patients with known conditions affecting urinary amino acid excretion. Reference intervals established for this method were also concordant with reference intervals obtained with IEC. Overall, aTRAQ reagents used in conjunction with LC-MS/MS should be considered a comparable alternative to IEC. The most attractive features of this methodology are the decreased run time and increased specificity.     

Determination of amino acids in human serum by capillary gas chromatography

A simple and rapid method for the determination of serum amino acids by gas chromatography (GC) has been developed. Following deproteinization of serum with perchloric acid, free amino acids in the supernatant were converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. All the derivatives of the 22 protein amino acids were completely resolved as single peaks within 9 min by GC. The calibration curves were linear in the range 0.2–50 μg of each amino acid, and the correlation coefficients were above 0.998. By using this method, serum amino acids could be directly analysed without prior clean-up procedure such as ion-exchange column chromatography except for deproteinization of the samples, and without any interference from coexisting substances. Overall recoveries of amino acids added to serum samples were 88–108%. Analytical results for serum amino acids from normal subjects are presented.     

A modified spray reagent for the detection of amino acids on thin layer chromatography plates

Summary. Identification of amino acids is extremely important for the evaluation of protein structure. Thin layer chromatography is an important tool for detecting amino acids by variety of spray reagents. Among these ninhydrin is the most popular due to its high sensitivity. However, ninhydrin produces the same purple/violet color with most amino acids. A spray reagent with high sensitivity for easy and rapid identification of amino acids on thin-layer plates has been introduced. Received March 14, 2000 Accepted August 31, 2000     

The use of a new silica gel separation system in thin-layer chromatography

Summary A new method for the chromatography of amino acids is described in which D- or L-amino acids are separated on ICT-Empore thin-layers. The compounds are developed ascending by means of normally used solvent systems. An overloading of the plates is nearly impossible. On the other hand, hydrophilic amino acids are well separated. A second front, moving with these amino acids and emerging with ninhydrin stain, was not detectable.     

Proteome analysis of Halobacterium sp. NRC-1 facilitated by the biomodule analysis tool BMSorter

To better understand the extremely halophilic archaeon Halobacterium species NRC-1, we analyzed its soluble proteome by two-dimensional liquid chromatography coupled to electrospray ionization tandem mass spectrometry. A total of 888 unique proteins were identified with a ProteinProphet probability (P) between 0.9 and 1.0. To evaluate the biochemical activities of the organism, the proteomic data were subjected to a biological network analysis using our BMSorter software. This allowed us to examine the proteins expressed in different biomodules and study the interactions between pertinent biomodules. Interestingly an integrated analysis of the enzymes in the amino acid metabolism and citrate cycle networks suggested that up to eight amino acids may be converted to oxaloacetate, fumarate, or oxoglutarate in the citrate cycle for energy production. In addition, glutamate and aspartate may be interconverted from other amino acids or synthesized from citrate cycle intermediates to meet the high demand for the acidic amino acids that are required to build the highly acidic proteome of the organism. Thus this study demonstrated that proteome analysis can provide useful information and help systems analyses of organisms.     

Analysis of phenylthiohydantoin amino acid mixtures for sequencing by thermospray liquid chromatography/mass spectrometry

Phenylthiohydantoin (PTH) amino acids, the derivatives of amino acids liberated in the course of automated N-terminal sequence analysis of peptides and proteins, are most commonly identified by high-performance liquid chromatography. This communication describes an extension to the methodology for PTH amino acid identification which exploits thermospray liquid chromatography/mass spectrometry for use in the confirmation of PTH amino acid identifications previously made solely on the basis of retention times. Thermospray mass spectra of the 19 synthetic PTH amino acids corresponding to the residues commonly observed during N-terminal sequencing have been acquired. These spectra show strong signals for the protonated molecular ion, accompanied in several cases by ions produced by limited fragmentation of the amino acid side chain and/or the PTH ring system. A reverse-phase separation protocol has been adapted for use with thermospray. The method permits recognition of the protonated molecular ions of all the standard PTH amino acids at the 150-pmol level on the basis of signal-to-noise ratios of 10:1 or better with full scanning. The method has been tested on the N-terminal amino acid sequence analysis of 200 pmol of the standard protein beta-lactoglobulin A, and has been found useful in the study of selected side-products of the sequencing chemistry.     

Mycosporine-like amino acids in the zooxanthella-ciliate symbiosis Maristentor dinoferus

Coral reef organisms living in mutualistic symbioses with phototrophic dinoflagellates are widespread in shallow UV-transparent waters. Maristentor dinoferus is a recently discovered species of marine benthic ciliate that hosts symbiotic dinoflagellates of the genus Symbiodinium. In this study, we tested this ciliate for the occurrence of mycosporine-like amino acids, a family of secondary metabolites that minimize damage from exposure to solar UV radiation by direct screening. Using high-performance liquid chromatography and liquid chromatography coupled to mass spectrometry, five mycosporine-like amino acids (shinorine, palythenic acid, palythine, mycosporine-2-glycine, and porphyra-334) were identified in aqueous methanolic extracts of the symbiosis. This is the first report of mycosporine-like amino acids in a marine ciliate.     

Determination of erythrocyte amino acids by gas chromatography

Erythrocyte amino acid levels were determined, by gas chromatography, in a group of 34 normal human adults. No significant sex or age correlations were noted.A method for the quantitative gas chromatographic analysis of free amino acids in erythrocytes is described. Following hemolysis and deproteinization the amino acids were isolated on a cation-exchange resin. Glutathione was removed from the amino acid mixture by adsorption on an anion-exchange resin. Following conversion to their N-acetyl-n-propyl esters, 19 amino acids were separated and quantitated by gas chromatography on a single column in 18 min. Typical reproducibility data indicate that a coefficient of variation of 2–5% is attainable.     

Analysis of amino acids in nectar from pitchers of Sarracenia purpurea (Sarraceniaceae)

Sarracenia purpurea L. (northern pitcher plant) is an insectivorous plant with extrafloral nectar that attracts insects to a water-filled pitfall trap. We identified and quantified the amino acids in extrafloral nectar produced by pitchers of S. purpurea. Nectar samples were collected from 32 pitchers using a wick-sampling technique. Samples were analyzed for amino acids with reverse-phase high-performance liquid chromatography with phenylisothiocyanate derivatization. Detectable amounts of amino acids were found in each of the 32 nectar samples tested. Mean number of amino acids in a nectar sample was 9 (SD = 2.2). No amino acid was detected in all 32 samples. Mean amount of amino acids in a nectar sample (i.e., amount per wick) was 351.4 ng (SD = 113.2). Nine amino acids occurred in 20 of the 32 samples (aspartic acid, cysteine, glutamic acid, glycine, histidine, hydroxyproline, methionine, serine, valine) averaging 263.4 ng (SD = 94.9), and accounting for ~75% of the total amino acid content. Nectar production may constitute a significant cost of carnivory since the nectar contains amino acids. However, some insects prefer nectar with amino acids and presence of amino acids may increase visitation and capture of insect prey.