Deriving the probabilities of water loss and ammonia loss for amino acids from tandem mass spectra

In protein identification through tandem mass spectrometry, it is critical to accurately predict the theoretical spectrum for a peptide sequence. The widely used prediction models, such as SEQUEST and MASCOT, ignore the intensity of the ions with important neutral losses, including water loss and ammonia loss. However, ignoring these neutral losses results in a significant deviation between the predicted theoretical spectrum and its experimental counterpart. Here, based on the "one peak, multiple explanations" observation, we proposed an expectation-maximization (EM) method to automatically learn the probabilities of water loss and ammonia loss for each amino acid. Then we employed these probabilities to design an improved statistical model for theoretical spectrum prediction. We implemented these methods and tested them on practical data. On a training set containing 1803 spectra, the experimental results show a good agreement with some known knowledge about neutral losses, such as the tendency of water loss from Asp, Glu, Ser, and Thr. Furthermore, on a testing set containing 941 spectra, the improved similarity between the experimental and predicted spectra demonstrates that this method can generate more reasonable predictions relative to the model that ignores neutral losses. As an application of the derived probabilities, we implemented a database searching method adopting the improved theoretical spectrum model with neutral loss ions estimated. Experimental results on Keller's data set demonstrate that this method can identify peptides more accurately than SEQUEST. In another application to validate SEQUEST's results, the reported peptide-spectrum pairs are reranked with respect to the similarity between experimental and predicted spectra. Experimental results on both LTQ and QSTAR data sets suggest that this reranking strategy can effectively distinguish the false negative predictions reported by SEQUEST.     

13N isotope studies on the pathway of ammonia assimilation in Bacillus megaterium and Escherichia coli.

The pathway of ammonia incorporation into amino acids was studied by use of 13N-ammonium ions in Bacillus megaterium and Escherichia coli that had been grown aerobically on a minimal salts medium containing NH4Cl as the source of nitrogen. Anion- and cation-exchange high-pressure-liquid chromatography was used to separate amino acids relevant to the several possible pathways for ammonia assimilation in bacteria. At an initial concentration of added NH4+ of 1 microM, the glutamine synthetase-glutamate synthase pathway represented the major pathway in both bacteria on the basis of the effects of inhibitors of that pathway (L-methionine-DL-sulfoximine and azaserine) and of transamination (aminooxy-acetate) and the observation that the specific activity of glutamine was greater initially than that of any other amino acid likely to be the first product of an assimilation pathway. The study provides (i) a new analytical method for 13N-tracer investigation of amino acids, (ii) confirmation of conclusions from enzymological studies on the pathway of ammonia assimilation in B. megaterium and E. coli, and (iii) proof that alanine dehydrogenase and aspartate ammonia lyase (aspartase) are not important pathways in B. megaterium at low NH4+ concentrations.     

Amino Acid Synthesis in Photosynthesizing Spinach Cells : EFFECTS OF AMMONIA ON POOL SIZES AND RATES OF LABELING FROM CO(2)

Isolated cells from leaves of Spinacia oleracea have been maintained in a state capable of high rates of photosynthetic CO₂ fixation for more than 60 hours. The incorporation of ¹⁴CO₂ under saturating CO₂ conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivity, and pool size have been determined as a function of time for most of the protein amino acids and for γ-aminobutyric acid. The measurements of specific radio-activities and of the approaches to ¹⁴C “saturation” of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids.     

Absorption of amino acids from the human mouth

Summary Certain amino acids were transported across buccal mucosa in vivo by a carrier-mediated process. Metabolic loss of L-amino acids from the mouth in a 5 min test period was negligible. The buccal mucosal transport process was stereospecific for most L-amino acids tested. The uptake of L-methionine and L-leucine showed a tendency to saturation with increasing substrate concentration. The absorption of L-leucine, L-isoleucine and L-methionine as single amino acids was inhibited in the presence of each other suggesting at least one common transport mechanism. Administration of equimolar amounts of amino acids revealed a specific pattern of absorption that could be classified into fast, intermediate, and slow groups. Absorption of some amino acids was at least partly dependent on the presence of sodium ions in the luminal solution. In conclusion, our studies demonstrate that the human buccal mucosa is permeable to L-amino acids in a selective manner, and may resemble absorption pattern similar to other locations of the gastrointestinal tract.This work was supported by Grant DK39147 from the National Institutes of Diseases and Digestive and Kidney Diseases, National Institutes of Health, United States Public Health Service, and The Lord Dowding Fund for Humane Research, London, U.K.     

The Universal Trend of Amino Acid Gain–Loss is Caused by CpG Hypermutability

Understanding the cause of the changes in the amino acid composition of proteins is essential for understanding the evolution of protein functions. Since the early 1970s, it has been known that the frequency of some amino acids in protein sequences is increasing and that of others is decreasing. Recently, it was found that the trends of amino acid changes were similar in 15 taxa representing Bacteria, Archaea, and Eukaryota. However, the cause of this similarity in the trend of the gains and losses of amino acids continued to be debated. Here, we show that this trend of the gain and loss of amino acids can be simply explained by CpG hypermutability. We found that the frequency of amino acids coded by codons with TpG dinucleotides and those with CpA dinucleotides is increasing, while that of amino acids coded by codons with CpG dinucleotides is decreasing. We also found that organisms that lack DNA methyltransferase show different trends of the gain and loss of amino acids. DNA methyltransferase methylates CpG dinucleotides and induces CpG hypermutability. The incorporation of CpG hypermutability into models of protein evolution will improve studies on protein evolution in different organisms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Neurochemical analysis of amino acids, polyamines and carboxylic acids: GC-MS quantitation of tBDMS derivatives using ammonia positive chemical ionization

The GC-MS quantitation of a large number of neurochemicals utilizing a single derivatization step is not common but is provided by the reagent N-(tert-butyldimethylsilyl)-N-methyltrifluro-acetamide (MTBSTFA). Previous workers have utilized this derivative for GC-MS analyses of amino acids, carboxylic acids and urea with electron impact (EI) and with positive chemical ionization (PCI; methane as reagent gas). However, these conditions yield significant fragmentation, decreasing sensitivity and in some cases reducing specificity for quantitation with selected ion monitoring (SIM). Additionally, the majority of studies have used a single internal standard to quantitate many compounds. In this study we demonstrate that using isotopic dilution combined with ammonia as the reagent gas for PCI analyses, results in high precision and sensitivity in analyzing complex neurochemical mixes. We also demonstrate for the first time the utility of this derivative for the analysis of brain polyamines and the dipeptide cysteinyl glycine. In the case of ammonia as the reagent gas, all amino acids, polyamines and urea yielded strong [MH](+) ions with little or no fragmentation. In the case of carboxylic acids, [M+18](+) ions predominated but [MH](+) ions were also noted. This approach was used to analyze superfusates from hippocampal brain slices and brain tissue extracts from brain lesion studies. The advantages of this methodology include: (i) simple sample preparation; (ii) a single derivatization step; (iii) direct GC-MS analysis of the reaction mix; (iv) high precision as a result of isotopic dilution analyses; (v) high sensitivity and specificity as a result of strong [MH](+) ions with ammonia reagent gas; (vi) no hydrolysis of glutamine to glutamate or asparagine to aspartate; and (vii) applicability to a wide range of neurochemicals.     

Nitrogen metabolism and excretion in the aquatic chinese soft-shelled turtle, Pelodiscus sinensis, exposed to a progressive increase in ambient salinity

This study aimed to determine effects of 6-day progressive increase in salinity from 1 per thousand to 15 per thousand on nitrogen metabolism and excretion in the soft-shelled turtle, Pelodiscus sinensis. For turtles exposed to 15 per thousand water on day 6, the plasma osmolality and concentrations of Na+, Cl- and urea increased significantly, which presumably decreased the osmotic loss of water. Simultaneously, there were significant increases in contents of urea, certain free amino acids (FAAs) and water-soluble proteins that were involved in cell volume regulation in various tissues. There was an apparent increase in proteolysis, releasing FAAs as osmolytes. In addition, there might be an increase in catabolism of certain amino acids, producing more ammonia. The excess ammonia was retained as indicated by a significant decrease in the rate of ammonia excretion on day 4 in 15 per thousand water, and a major portion of it was converted to urea. The rate of urea synthesis increased 1.4-fold during the 6-day period, although the capacity of the hepatic ornithine urea cycle remained unchanged. Urea was retained for osmoregulation because there was a significant decrease in urea excretion on day 4. Increased protein degradation and urea synthesis implies greater metabolic demands, and indeed turtles exposed to 15 per thousand water had significantly higher O2 consumption rate than the freshwater (FW) control. When turtles were returned from 15 per thousand water to FW on day 7, there were significant increases in ammonia (probably released through increased amino acid catabolism) and urea excretion, confirming that FAAs and urea were retained for osmoregulatory purposes in brackish water.     

Effects of impermeant medium ions on the composition of rabbit renal cortical slices

When incubated in isosmotic oxygenated medium in which chloride was completely replaced by gluconate, rabbit renal cortical slices lost chloride with sodium, potassium and water before reaching a new steady-state composition after 15-30 min. When corrected for extracellular space, there was an electroneutral loss of alkali metal cations (Na + K) with chloride, accompanied by isosmotic loss of water from the cells. The losses of chloride and water were independent of medium pH over the range of 6.4-8.2, and were the same with potassium rather than sodium as the dominant medium cation. Incubation in isosmotic sodium chloride medium restored tissue composition of slices transferred from gluconate medium. This recovery was not dependent specifically upon medium chloride, for slice water content also recovered when nitrate rather than chloride was substituted for medium gluconate. With sodium completely replaced by n-methyl d-glucamine (nmdG+), cells in slices lost far more sodium and potassium than chloride before reaching a new steady-state composition after some 30 min. However, the loss of water was as predicted from the total losses of measured inorganic ions. With sodium and chloride completely replaced by nmdG+ and gluconate, there was a greater loss of water than found with unilateral substitutions. Again, the combined loss of diffusible inorganic cations exceeded the loss of chloride but the water loss was that expected for isosmotic loss accompanying the measured losses of ions. These results reveal that both gluconate and nmdG+ behave as impermeant ions in this tissue preparation. It is suggested that, in the absence of medium sodium, sodium-hydrogen exchange is inhibited. Retained hydrogen ions are buffered on charged cellular non-diffusible solutes and the associated hydroxyl (or bicarbonate) ions are lost from the cells accompanied by the inorganic univalent cations lost in excess of chloride in nmdG+ medium.     

Mechanisms of ammonia and ammonium ion toxicity in animal cells: Transport across cell membranes

A model for transport of ammonia and ammonium ions across cell membranes is presented. The model suggests that ammonium ions compete with potassium ions for inward transport, over the cytoplasmic membrane, via potassium transport proteins like the Na⁺/K⁺-ATPase and the Na⁺K⁺2Cl⁻-cotransporter. It also explains the difference between the ammonia/ammonium that is added to the cells and which is formed by the cells during metabolism of amino acids, especially glutamine and glutamate. The ammonium transport and subsequent events lead to predictable intracellular and extracellular pH (pH_e) changes. Experiments which verified the model and the predicted consequences were performed by measurements of the pH_e in concentrated cell suspensions. Addition of ammonium ions caused a time-dependent pH_e increase which was inhibited by potassium ions. The test system is not per se specific for transport measurements but the effect of potassium ions on the pH_e strongly favors our suggested model. Simple diffusion of ammonium ions would not be counteracted by potassium ions. The results show that ammonium ion transport in the murine myeloma cell line (Sp2/0-Ag14) used is inhibited by an excess of potassium ions. Results from experiments with specific inhibitors of suggested transport proteins were not conclusive. It is postulated that one important toxic effect of ammonia/ammonium is an increased demand for maintenance energy, caused by the need to maintain ion gradients over the cytoplasmic membrane. The results also suggest that potassium ions can be used to detoxify ammonia/ammonium in animal cell cultivations.     

Nitrogenous fertilizer transformations in the sudan Gezira soil

Summary and Conclusions Direct measurements were made of losses of ammonia during the transformation of urea and ammonium sulphate, surface-applied to alkaline Gezira soil in containers incubated in the field, under different rates of nitrogen application and moisture conditions.The highest rate of ammonia loss occurred during the first week after application with both fertilizers, thereafter decreasing to lower values. The cumulative ammonia loss was higher with higher application of nitrogen. Ammonium sulphate gave consistently higher ammonia losses than urea and losses from open soil system were generally less than from soil in polythene bags.With lowest irrigation level used, ammonia loss attained a sizeable value throughout the incubation period with both fertilizers. With the higher moisture levels, the magnitude of ammonia loss decreased appreciably, much more so with urea than with ammonium sulphate. Induced drying and rewetting prolonged the duration of loss and increased the magnitude of cumulative loss. An appreciable loss of ammonia may take place from fertillzed Gezira Soil under warm conditions, low moisture levels and high fertilizer concentration; this may be the case with patchy fertilizer distribution and frequent light showers during early summer. It is advisable to apply the urea or ammonium sulphate when conditions are most favourable for nitrification.     

Effect of fasting on urinary excretion of water and nitrogen in the rat

Rats housed in metabolic cages undergoing 48 h fasting showed reduced daily intake of drinking water and increased urinary volume. Both the daily amount of urinary creatinine and urea excreted were diminished with fasting, while that of ammonia was augmented. Besides demonstrating metabolic water loss under fasting, results suggest that ammonia derived from glutamine in the kidney, rather than urea formed in the liver, is the main nitrogenous catabolic product of amino acids in fasting.     

How many Na+-dependent carriers for L-alanine and L-proline in the eel intestine? Studies with brush-border membrane vesicles

Using brush-border membrane (BBM) vesicles prepared from the intestine of the European eel, the specificity of L-alanine and L-proline Na+-dependent transport was investigated by measuring the uptake of isotopically labelled substrates. In the presence of Na+ ions, cross-inhibition between alanine and proline transports was observed; in addition alpha-(methylamino)isobutyric acid (MeAIB) inhibited proline but had no effect on alanine uptake. These results can be explained by the presence, in eel intestinal BBM vesicles, of at least two distinct agencies for Na+-dependent proline and alanine translocation. The first system is specific for alanine and short-chain neutral amino acids; the second system, specific for imino acids and the N-methylated analogues, is regulated by alanine concentration.     

The mechanisms of amino acids synthesis by high temperature shock-waves.

The mechanisms of amino acids synthesis behind high temperature shock-waves were elucidated and distinction was made between the steps occurring in the gas phase and those occurring in solution. In the presence of water vapor, aldehydes and HCN are formed separately in regions of different temperatures along the reacting gas. The aldehydes and ammonia condense to aldimines which add HCN to form alpha-amino nitriles, all in the gas phase. The hydrolysis to amino acids takes place in solution. In the absence of water vapor, aldimines and HCN are formed in the gas phase but condense to alpha-amino nitriles only in solution. A fair amount of oxygen only lowers the production of amino acids, which consequently could be still produced in the presence of oxygen in the Earth's primitive atmoshere. The waterless mechanism can operate in the Jovian atmosphere and supply it with ample amounts of amino acids, especially aspartic.     

Ammonia volatilization after injection of anhydrous ammonia into arable soils of different moisture levels

Laboratory experiments have shown appreciable losses of ammonia after injection of anhydrous ammonia into dry and wet soils. In this study losses of ammonia injected into a moist (tension 10 kPa), dry (tension 160 kPa) and a wet (tension 1.6 kPa) sandy loam were measured under field conditions using wind tunnels. Losses were insignificant from a moist soil. However losses from a dry and a wet soil were 20% and 50% of injected ammonia, respectively. From the dry soil, losses of gaseous ammonia took place within the first hours after injection, which indicates a rapid transport through cracks and voids. From the wet soil, 20% of the injected ammonia was lost more gradually between 6 h and 6 d. This indicates that upward movement of water due to evaporation may be the cause of these ammonia losses which proceeded for longer periods.     

Glucose-induced Release of Amino Acids from Saccharomyces carlsbergensis by Action on the Cytoplasmic Membrane

When washed yeast cells grown under appropriate conditions were suspended in glucose solution there was a sudden release of α-amino nitrogen into the medium. This released material was of low molecular weight, and its composition was closely similar to that of the intracellular free amino acid pool. During the leakage of amino acids, the yeast did not efficiently absorb labeled amino acids added to the test medium, despite the rapid uptake and metabolism of glucose. Uptake of a labeled amino acid and reabsorption of the released α-amino nitrogen occurred almost simultaneously. When these yeast cells were exposed to glucose in the presence of calcium ions, leakage was strongly inhibited. Butanol under the same conditions increased glucose-induced leakage of cell contents. The adenosine triphosphatase activity of intact yeast cells exposed to glucose was greater than that of cells exposed to water. Yeast cells treated with glucose prior to equilibration with sorbose exhibited less ability to retain the sorbose when washed at 0 C than did cells pretreated with water. It was concluded that glucose-induced leakage of amino acids was the result of two factors acting together. These were (i) a change in membrane permeability associated with glucose uptake, and (ii) a temporary shortage of energy for amino acid uptake or retention.     

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.     

Carbamyl phosphate and citrulline formation by phototrophic bacteria

The purple bacteria Ectothiorhodospira shaposhnikovii and Rhodospirillum rubrum are capable of synthesizing citrulline in the presence of ammonium hydrocarbonate, ornithine, ATP and Mg2+ ions. Citrulline biosynthesis by these phototrophic bacteria is presumed to be catalysed by carbamate kinase and ornithine transcarbamoylase. Therefore, certain phototrophic bacteria can assimilate carbon dioxide and ammonia for biosynthesis of amino acids, in particular, citrulline and, apparently, arginine.     

The simultaneous quantitation of ten amino acids in soil extracts by mass fragmentography

A specific and sensitive method for the identification and simultaneous quantitation by mass fragmentography of 10 of the amino acids present in soil has been developed. The technique uses a computer-driven quadrupole mass spectrometer, and a commercial preparation of deuterated amino acids is used as internal standard for purposes of quantitation. The results obtained are comparable with those from an amino acid analyser. In the quadrupole mass spectrometer-computer system used, up to 25 preselected ions may be monitored sequentially. This allows a maximum of 12 different amino acids (one specific ion in each of the undeuterated and deuterated amino acid spectra) to be quantitated. The method is relatively rapid (analysis time of approximately 1 hr) and is capable of the quantitation of nanogram quantities of amino acids.     

The crystal structure at 2A resolution of the Ca2+ -binding protein S100P

S100P is a small calcium-binding protein of the S100 EF-hand-containing family of proteins. Elevated levels of its mRNA are reported to be associated with the progression to hormone independence and metastasis of prostate cancer and to be associated with loss of senescence in human breast epithelial cells in vitro. The first structure of human recombinant S100P in calcium-bound form is now reported at 2.0A resolution by X-ray diffraction. A flexible linker connects the two EF-hand motifs. The protein exists as a homodimer formed by non-covalent interactions between large hydrophobic areas on monomeric S100P. Experiments with an optical biosensor to study binding parameters of the S100P monomer interaction showed that the association rate constant was faster in the presence of calcium than in their absence, whereas the dissociation rate constant was independent of calcium. The K(d) values were 64(+/-24)nM and 2.5(+/-0.8) microM in the presence and in the absence of calcium ions, respectively. Dimerization of S100P is demonstrated in vivo using the yeast two-hybrid system. The effect of mutation of specific amino acids suggests that dimerization in vivo can be affected by amino acids on the dimer interface and in the hydrophobic core.     

Characterization of amino acid transport in membrane vesicles from the thermophilic fermentative bacterium Clostridium fervidus.

Amino acid transport was studied in membrane vesicles of the thermophilic anaerobic bacterium Clostridium fervidus. Neutral, acidic, and basic as well as aromatic amino acids were transported at 40 degrees C upon the imposition of an artificial membrane potential (delta psi) and a chemical gradient of sodium ions (delta microNa+). The presence of sodium ions was essential for the uptake of amino acids, and imposition of a chemical gradient of sodium ions alone was sufficient to drive amino acid uptake, indicating that amino acids are symported with sodium ions instead of with protons. Lithium ions, but no other cations tested, could replace sodium ions in serine transport. The transient character of artificial membrane potentials, especially at higher temperatures, severely limits their applicability for more detailed studies of a specific transport system. To obtain a constant proton motive force, the thermostable and thermoactive primary proton pump cytochrome c oxidase from Bacillus stearothermophilus was incorporated into membrane vesicles of C. fervidus. Serine transport could be driven by a membrane potential generated by the proton pump. Interconversion of the pH gradient into a sodium gradient by the ionophore monensin stimulated serine uptake. The serine carrier had a high affinity for serine (Kt = 10 microM) and a low affinity for sodium ions (apparent Kt = 2.5 mM). The mechanistic Na+-serine stoichiometry was determined to be 1:1 from the steady-state levels of the proton motive force, sodium gradient, and serine uptake. A 1:1 stoichiometry was also found for Na+-glutamate transport, and uptake of glutamate appeared to be an electroneutral process.