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.     

不同来源仙草的氨基酸比较分析

采用盐酸水解法分析不同来源仙草的氨基酸组成及含量差异,结果表明供试仙草均含有17种氨基酸和7种必需氨基酸,氨基酸总量为4.75％～13.65％,17种氨基酸总量和7种必需氨基酸总量的高低顺序都是9＞10＞5＞8＞1 ＞6＞4＞3＞2＞7,均以9号仙草含量最高,7号仙草最低.各种氨基酸含量高低顺序相似,以谷氨酸、天门冬氨...     

A Simple Gasometric Method for Determination of Free Amino Acid Contents in Biological Fluids

A simple method for the determination of total amino acid contents in biological fluids which contain various amino acids, peptides and proteins is described. This method is based on the determination in a Gilson differential respirometer of CO₂ gas evolved by the reaction of chloramin-T with α-amino-carboxylic acids in 5% trichloroacetic acid. The use of the trichloroacetic acid greatly suppressed interference by amines and ammonia, thus making this method appreciable to the determination of free amino acids in biological fluids without prior separation. Samples containing 1~20 μmoles of free amino acids can be assayed with accuracy of ±3%. This method was applied to the study of proteolytic digestion of casein.     

A precise method for the quantitation of proteins taking into account their amino acid composition.

A method for the quantitation of protein in biological material is described which gives the same response for all proteins irrespective of their amino acid composition. The method is based on the ninhydrin reaction of amino acids released after total acid hydrolysis of 5- to 20-μl solutions containing 1 to 100 μg of protein. The ammonia is released from the hydrolysate by diffusion and the amino acids are quantitated without fractionation using the continuous-flow system of an amino acid analyzer. Calibration is obtained with solutions of known amino acid content. The protein of a sample is calculated by multiplying the nanomoles of total amino acids found by a conversion factor F. F is the weight in micrograms of 1 nmol of the specific mixture of amino acid residues that the protein of the sample is composed of F has to be determined once for all further quantitations of the same material by quantitative amino acid analysis following standard procedures. By this method as little as 30 ng of protein per aliquot of hydrolysate analyzed can be determined.     

Amino acid requirements in humans: with a special emphasis on the metabolic availability of amino acids

Due to advances made in the development of stable isotope based carbon oxidation methods, the determination of amino acid requirements in humans has been an active area of research for the past 2 decades. The indicator amino acid oxidation (IAAO) method developed in our laboratory for humans has been systematically applied to determine almost all indispensable amino acid requirements in adult humans. Nutritional application of experimentally derived amino acid requirement estimates depends upon the capacity of food proteins to meet the amino acid requirements in humans. Therefore, there is a need to know the proportion of dietary amino acids which are bioavailable, or metabolically available to the body for protein synthesis following digestion and absorption. Although this concept is widely applied in animal nutrition, it has not been applied to human nutrition due to lack of data. We developed a new in vivo method in growing pigs to identify the metabolic availability of amino acids in foods using the IAAO concept. This metabolic availability method has recently been adapted for use in humans. As this newly developed IAAO based method to determine metabolic availability of amino acids in foods is suitable for rapid and routine analysis in humans, it is a major step forward in defining the protein quality of food sources and integrating amino acid requirement data with dietary amino acid availability of foods.     

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.     

Separation of D- and L-amino acids by ion exchange column chromatography in the form of alanyl dipeptides

Summary A method of ion exchange column chromatography was developed for the determination of D- and L-amino acids in the form of diastereomeric dipeptide. First the protein containing samples were hydrolyzed with 6 molar hydrochloric acid, then the single amino acids were separated in an LKB automated amino acid analyzer with the LKB fraction collector. Following lyophilization, the single amino acids were transformed into alanyl dipeptides with tertiary-butyloxycarbonil-L-alanine-N-hydroxy-succinimide (t-BOC-L-Ala-ONSu) active ester. The alanyl dipeptides were easily separated from one another and the initial amino acids. Determination of the D- and L-amino acids in this form is relatively accurate and reproducible but takes some time (33–38 min). Accuracy of the determination is satisfactory. The coefficient of variation amounts to 3–5%. The use of the method is suggested to laboratories having an amino acid analyzer and wish to determine D-and L-amino acids in synthetic-amino acids complements, peptides or natural materials.     

Measurement of total protein in plant samples in the presence of tannins

A method for measuring total protein in situ in plant samples has been developed using the determination of amino acids released by acid hydrolysis of dried plant material. Standard proteins and plant samples were hydrolyzed with 3% sulfuric acid at 100 degrees C for 24 h and the amino acids released were measured with ninhydrin. Unhydrolyzed plant extracts were also analyzed for free amino acids with ninhydrin. Total amino acid equivalents (protein plus free amino acids) of a diverse set of plant samples was significantly correlated with total protein as estimated by elemental analysis (N X 6.25). The Lowry method as modified by precipitation of proteins with trichloroacetic acid was found to be unsatisfactory for dried plant samples due to the incomplete extractability of proteins. Although some alkaloids caused increased absorbance with ninhydrin, interference with quantification of protein is likely to be minimal. Tannins interfered with the Lowry and Bradford methods but not the ninhydrin method.     

Determination of amino acids in different regions of the rat brain. Application to the acute effects of tetrahydrocannabinol (THC) and trimethyltin (TMT)

A modified HPLC method is described for the determination of amino acids [aspartic acid, glutamic acid, glutamine, glycine, taurine, and gamma-aminobutyric acid (GABA)] in brain tissue utilizing precolumn derivatization with o-phthalaldehyde (OPA)-tert-butyl-thiol and electrochemical detection. A simple extraction procedure was employed and DL-homoserine used as internal standard. A neurotoxin previously shown to affect brain amino acids (trimethyltin, TMT) and a psychoactive compound hypothesized to act on these neurochemicals (delta-9-tetrahydrocannabinol, THC) were administered to adult male rats and amino acids were measured. Results revealed a gradient of distribution of most amino acids, with lowest levels posteriorly in the brain stem and increasing to the highest values in anterior cortical regions. TMT increased glutamine significantly in all brain regions examined, but increased glycine and decreased taurine only in the frontal cortex and hippocampus. No significant changes in any amino acid were found in hippocampus after THC treatment. The results establish the validity and usefulness of this HPLC method for detecting neurotoxicity-related changes in brain amino acid metabolism.     

Analysis of all protein amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography

A gas chromatographic method for the separation and quantitation of the 20 protein amino acids is described using N-methyl-N(tert.-butyldimethylsilyl)trifluoroacetamide, with 1% tert.-butyldimethylchlorosilane as catalyst, to prepare the tert.-butyldimethylsilyl amino acid derivatives. Alkylsilylation of amino acids proceeds at 140 degrees C in 20 min. The derivatives formed in the one-step reaction are used directly for gas-liquid chromatographic analysis, using a flame-ionization detector, without prior isolation or purification. Complete separation and quantitation of all protein amino acids are readily achieved using a 15-m DB-5 capillary column. Strict linearity extends from less than 15 to about 100 ng for all amino acids except Arg, which has a linear range from 50 to 300 ng. The limits of detection, however, range from one to several hundred nanograms. The method was used to analyze the free amino acid pool in carnation petals.     

Characterization of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae

Intact vacuoles are released from spheroplasts of Saccharomyces cerevisiae by means of a gentle mechanical disintegration method. They are purified by centrifugation in isotonic density gradients (flotation and subsequent sedimentation), and analyzed for their soluble amino acid content. The results indicate that about 60% of the total amino acid pool of spheroplasts is contained in the vacuoles. This may be an underestimate, as it presupposes no loss of amino acids from the vacuoles during the purification procedure. The amino acid concentration in the vecuoles is calculated to be approximately 5 times that in the cytoplasm if the total volumes of the two compartments are used for the calculation. The vacuolar amino acid pool is rich in basic amino acids, and in citrulline and glutamine, but contains a remarkably small amount of glutamate. Radioactive labeling experiments with spheroplasts indicate that the vacuolar amino acids are separated from the metabolically active pools located in the cytoplasm. This is particularly evident for the basic amino acids and glutamine; in contrast, the neutral amino acids and glutamate appear to exchange more rapidly between the cytoplasmic and the vacuolar compartments of the cells.     

Measurement of free amino acid levels in ultrafiltrates of blood plasma by high-performance liquid chromatography with automatic pre-column derivatization

Although there are many techniques available for the analysis of amino acids, deproteinization is still one of the major problems in the analysis of amino acids in physiological fluids. The method used to prepare the plasma and to remove the plasma protein has a marked effect on the final results. The most widely used method of deproteinization is precipitation with 5-sulphosalicylic acid followed by centrifugation to remove the precipitated protein. We have not had success in using this deproteinization agent for the analysis of plasma amino acids by a high-performance liquid chromatographic method with automatic pre-column o-phthaldialdehyde—3-mercaptopropionic acid and 9-fluorenylmethyl chloroformate derivatization because of the adverse effect of the sulphosalicyclic acid supernatant on the quantitation and separation. Ultrafiltration was used as an alternative method for the preparation of plasma samples in this experiment. The results were satisfactory for the analysis of plasma amino acids in 1500 samples during a period of four years. Some factors that might influence the results of the ultrafiltration were investigated.     

Direct extract derivatization for determination of amino acids in human urine by gas chromatography and mass spectrometry

The purpose of this study was to develop a simple and accurate analytical method to determine amino acids in urine samples. The developed method involves the employment of an extract derivatization technique together with gas chromatography-mass spectrometry (GC-MS). Urine samples (300 microl) and an internal standard (10 microl) were placed in a screw tube. Ethylchloroformate (50 microl), methanol-pyridine (500 microl, 4:1, v/v) and chloroform (1 ml) were added to the tube. The organic layer (1 microl) was injected to a GC-MS system. In this proposed method, the amino acids in urine were derivatized during an extraction, and the analytes were then injected to GC-MS without an evaporation of the organic solvent extracted. Sample preparation was only required for ca. 5 min. The 15 amino acids (alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tyrosine, tryptophan, valine) quantitatively determined in this proposed method. However, threonine, serine, asparagine, glutamine, arginine were not derivatized using any tested derivatizing reagent. The calibration curves showed linearity in the range of 1.0-300 microg/ml for each amino acid in urine. The correlation coefficients of the calibration curves of the tested amino acids were from 0.966 to 0.998. The limit of detection in urine was 0.5 microg/ml except for aspartic acid. This proposed method demonstrated substantial accuracy for detection of normal levels. This proposed method was limited for the determination of 15 amino acids in urine. However, the sample preparation was simple and rapid, and this method is suitable for a routine analysis of amino acids in urine.     

Quantitative identification of N-terminal amino acids in proteins by radiolabeled reductive methylation and amino acid analysis: application to human erythrocyte acetylcholinesterase

A novel method of determining N-terminal amino acids in proteins is introduced. Reductive methylation of a protein with radiolabeled formaldehyde methylates both the alpha-amino group of the N-terminal amino acid and the epsilon-amino groups of Lys residues. The radiomethylated amino acids are stable to acid hydrolysis, and each of 16 possible hydrolysis-stable N-terminal amino acids can be identified by the unique elution positions of its N alpha-methyl and N alpha,N alpha-dimethyl derivatives with an appropriate amino acid analyzer elution schedule. The technique is at least as sensitive as other N-terminal amino acid determinations and, in addition, permits a quantitative evaluation of the number of N-terminal groups in a sample. Reductive methylation of bovine serum albumin revealed N-terminal Asp at a stoichiometry of 0.97 amino acid residue per polypeptide, while methylation of prolactin resulted in 0.86 residue of N-terminal Thr per polypeptide. Human erythrocyte acetylcholinesterase contained two N-terminal amino acids with stoichiometries of 0.66 Glu and 0.34 Arg per 70-kDa subunit. Identification of Glu as the principal N-terminus of acetylcholinesterase was confirmed by Edman sequencing.     

Phosphorus and nitrogen co-limitation of forest ground vegetation under elevated anthropogenic nitrogen deposition

Amino acid nitrogen isotopic analysis is a relatively new method for estimating trophic position. It uses the isotopic difference between an individual’s ‘trophic’ and ‘source’ amino acids to determine its trophic position. So far, there is no accepted explanation for the mechanism by which the isotopic signals in ‘trophic’ and ‘source’ amino acids arise. Yet without a metabolic understanding, the utility of nitrogen isotopic analyses as a method for probing trophic relations, at either bulk tissue or amino acid level, is limited. I draw on isotopic tracer studies of protein metabolism, together with a consideration of amino acid metabolic pathways, to suggest that the ‘trophic’/‘source’ groupings have a fundamental metabolic origin, to do with the cycling of amino-nitrogen between amino acids. ‘Trophic’ amino acids are those whose amino-nitrogens are interchangeable, part of a metabolic amino-nitrogen pool, and ‘source’ amino acids are those whose amino-nitrogens are not interchangeable with the metabolic pool. Nitrogen isotopic values of ‘trophic’ amino acids will reflect an averaged isotopic signal of all such dietary amino acids, offset by the integrated effect of isotopic fractionation from nitrogen cycling, and modulated by metabolic and physiological effects. Isotopic values of ‘source’ amino acids will be more closely linked to those of equivalent dietary amino acids, but also modulated by metabolism and physiology. The complexity of nitrogen cycling suggests that a single identifiable value for ‘trophic discrimination factors’ is unlikely to exist. Greater consideration of physiology and metabolism should help in better understanding observed patterns in nitrogen isotopic values.     

Selective separation of amino acids by reactive extraction

The method of reactive extraction with di-(2-ethylhexyl)phosphoric acid (D2EHPA) for the separation of a range of amino acids is studied. The results obtained on the individual reactive extraction indicated the possibility of the amino acids selective separation as a function of the pH value of aqueous solution and the acidic or basic character of each amino acid. Thus, using multistage extraction, the total separation of the following amino acids groups has been performed: neutral amino acids (l-glycine, l-alanine, l-tryptophan) at pH 5–5.5 (nine extraction stages), basic amino acids (l-lysine, l-arginine) and l-cysteine at pH 4–4.5 (ten extraction stages), l-histidine at pH 3–3.5 (five extraction stages), and acidic amino acids (l-aspartic acid, l-glutamic acid) at pH 2–2.5 (three extraction stages).     

Peptide libraries: Determination of relative reaction rates of protected amino acids in competitive couplings

In the solid phase preparation of synthetic peptide libraries, equimolarity of the resultant peptides in the mixture simplifies the identification of active compounds. Two primary methods for the preparation of combinatorial peptide mixtures are currently used. In the first method, the starting resin is divided into equal aliquots, individual amino acids are coupled to each aliquot, and the resin is then recombined. This process is repeated for each position. However, due to the physical process, each resin bead contains only one peptide sequence. Statistically, for mixtures of longer sequences, an ever-increasing amount of resin is necessary to ensure complete representation of each peptide in the library. Thus, each peptide will be represented in the library if a sufficient number of resin beads are used. In addition, the concentration of each peptide in the library depends on both the number of mixture positions in the library and the amount of resin used. In the second method, mixtures of amino acids are coupled simultaneously at each addition step. The proportion of each amino acid in the reaction mixture is varied inversely to its reaction rate such that, ideally, an equimolar mixture of each peptide is synthesized. An advantage of this method over the previous method is that each peptide is ensured to be represented in the library, although not necessarily in equimolar amounts. It is known that not only do the coupling rates of each amino acid vary, but the coupling rates of individual amino acids also change when coupled to different amino acid resins. Consequently, in order to obtain equimolar peptide mixtures through the use of mixtures of protected amino acids, the ratio of reaction rates of one amino acid over another must be constant irrespective of the resin-bound amino acid. If this premise is true, this method of synthesis offers a significant advantage over the previous method since, theoretically, equimolar peptide libraries could be synthesized. The influence of the resin-bound amino acid on the relative reaction rates of incoming amino acids was investigated in the current study. © 1994 John Wiley & Sons, Inc.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based amino acid analysis

Amino acid analysis has been an integral part of analytical biochemistry for more than 50 years. However, its experimental design, which includes derivatization of amino acids followed by some kind of chromatographic separation, has not changed over the years. We have developed a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-based method for the quantitative analysis of amino acids. This method does not require any amino acid modification, derivatization, or chromatographic separation. The data acquisition time is decreased to several seconds for a single sample. No significant ion suppression effects were observed with the developed sample deposition technique, and the method was found to be reproducible. Linear responses between the amino acid concentration and the peak intensities ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300 microM, and correlation coefficients were between 0.983 (for arginine) and 0.999 (for phenylalanine). Limits of quantitation were between 0.03 microM (for arginine) and 3.7 microM (for histidine and homocysteine). This method was applicable to the mixtures of free amino acids as well as to HCl hydrolysates of proteins. Furthermore, we have shown that this method can be applied to other biologically important low-molecular weight compounds such as glucose.     

Dansylated amino acid separation by polyacrylamide gel electrophoresis

The paper describes a method for separation of dansylated amino acids by polyacrylamide gel electrophoresis. The methods allows a simultaneous analysis of 20-30 samples. The sensitivity of the method is 1 x 10(-9)-1 x 10(-10) M amino acid. The method permits separation of all amino acids formed during acid hydrolysis of proteins except for two pairs: Ile, Phe and Val, Asp.     

Optimizing the protein nutrition of growing-finishing pigs

Growing-finishing pigs should consume each day the minimum amounts of energy and amino acids needed for maximum lean deposition. This should optimize performance traits, carcass leanness, and N excretion. These ideal conditions are difficult to achieve under experimental or farm conditions due to the factors affecting amino acid requirements and feed intake on a daily basis. Lean deposition rate and sex are two of the major factors affecting amino acid needs. If possible, maximum lean deposition rates should be determined for each herd in order to customize feeding programs, and split-sex feeding will improve N utilization.

Amino acid requirements have been determined empirically and by the factorial method. The latter is preferred if the efficiency of use of absorbed amino acids can be accurately determined. Development of computer models will likely be needed to accomplish this. Apparent ileal digestibility of amino acids is the most practical means of estimating amino acid absorption at present, although it likely overestimates amino acid availability for some amino acids.

Crystalline amino acids can be used to improve amino acid balance and reduce excessive intake of protein which should improve feed efficiency. A portion of the high-quality protein feeds in pig diets can be replaced by synthetic amino acids without sacrificing performance, but the effects of these substitutions on carcass merit is uncertain.

Excretion of N, and the concomitant reduction of N in manure that has to be disposed of, can be manipulated nutritionally by increased use of crystalline amino acids to lower dietary protein, by use of highly digestible feedstuffs and by precise matching of amino acid needs to amino acid supply. Use of these factors could lead to a reduction in total N wastes of 20–30%.