In vitro quantification of hydrolysis-induced racemization of amino acid enantiomers in environmental samples using deuterium labeling and electron-impact ionization mass spectrometry

D-amino acids indicate aging, bacterial origin, and pathogenic properties of peptides in the environment, but the reliable assessment of D-enantiomers must account for a yet unknown formation during hydrolyses. Here, we introduce a method for the in vitro determination of the hydrolysis-induced racemization (HIR) of amino acids in environmental samples. It involves hydrolyses with hydro- and deuteriochloric acid (6 M, 12 h, 105 degrees C), desalting, and selective detection of chiral mass fragments of amino acid-N-pentafluoropropionyl derivatives. D-Amino acids formed in 2HCl incorporated deuterium into their C(alpha) position. This resulted in a relative signal loss of the nondeuterated fragment compared with the 1HCl hydrolysate. Mathematically evaluating the relative target signal intensities of both hydrolysates allowed the quantification of the proportion of D-amino acids formed during sample processing. Side-chain incorporations of deuterium were no limitations for this method as they could be estimated from that of the respective L-enantiomers. In soil and litter samples, between 0 (D-glutamic acid) and 85% (D-alloisoleucine) of the detected D-amino acids were formed upon hydrolysis (standard error, 5-11%). For a given amino acid, the HIR varied by a factor of 2-10 between samples, thereby confirming that HIR must be individually assessed for samples from different environments.     

Development of an amino acid sequence and D/L-configuration determination method of peptide with a new fluorescence Edman reagent, 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate

On the basis of the relationship between the fluorescence characteristics of the benzofurazan compounds and the Hammett constants (sigma p), a new fluorescence Edman reagent, 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MTBD-NCS) was designed and synthesized. MTBD-thiohydantoin (TH)-amino acid derivatives produced by the Edman sequencing method gave fluorescence, whereas other degradation byproducts such as MTBD-thiocarbamoyl (TC)- or carbamoyl (CA)-amino acids did not fluoresce. MTBD-NCS was applicable as an Edman sequencing reagent to the simultaneous determination of both the sequence and D/L-configuration of amino acids in peptides. Boron trifluoride (BF3) and HC1/methanol were adopted as the cyclization/cleavage and conversion reagents to suppress the amino acid residue racemization. The MTBD-TH-amino acids were separated on a reversed-phase column for amino acid sequencing, and their enantiomers were resolved on two types of polysaccharide-based chiral stationary phases for D/L-configuration determination. The method was successfully applied to the sequence and D/L-configuration determination of D-amino acid-containing peptide [D-Ala2]-deltorphin II.     

Determination of aromatic and sulfur-containing amino acids, peptides, and proteins using high-performance liquid chromatography with photolytic electrochemical detection

Aromatic amino acids, sulfur-containing amino acids, peptides containing such constituents, and proteins can now be detected in high-performance liquid chromatography by the use of on-line, postcolumn, continuous photolytic derivatization with electrochemical (HPLC-hv-EC) detection. The overall approach is a very simple, reproducible, rapid, and fully automatable approach for the determination of certain amino acids, peptides, and proteins with excellent selectivity, sensitivity, and linearities of response. Dual-electrode response ratios, lamp-on/lamp-off behavior, and chromatographic capacity factors all contribute to the enhanced selectivity of the overall HPLC-hv-EC determination for these particular classes of bioorganics and biopolymers. The analytical figures of merit, chromatography detection, and method validation approaches have all been optimally derived and demonstrated reproducible. Applications of the basic methodology to real-world samples are demonstrated and validated.     

Chiral recognition of amino acids by use of a fluorescent resorcinarene

The spectroscopic properties of a chiral boronic acid based resorcinarene macrocycle employed for chiral analysis were investigated. Specifically, the emission and excitation characteristics of tetraarylboronate resorcinarene macrocycle (TBRM) and its quantum yield were evaluated. The chiral selector TBRM was investigated as a chiral reagent for the enantiomeric discrimination of amino acids using steady-state fluorescence spectroscopy. Chiral recognition of amino acids in the presence of the macrocycle was based on diastereomeric complexes. Results demonstrated that TBRM had better chiral discrimination ability for lysine as compared to the other amino acids. Partial least squares regression modeling (PLS-1) of spectral data for macrocycle-lysine guest-host complexes was used to correlate the changes in the fluorescence emission for a set of calibration samples consisting of TBRM in the presence of varying enantiomeric compositions of lysine. In addition, validation studies were performed using an independently prepared set of samples with different enantiomeric compositions of lysine. The results of multivariate regression modeling indicated good prediction ability of lysine, which was confirmed by a root mean square percent relative error (RMS%RE) of 5.8%.     

Synergistic effects on enantioselectivity of zwitterionic chiral stationary phases for separations of chiral acids, bases, and amino acids by HPLC

In an attempt to overcome the limited applicability scope of earlier proposed Cinchona alkaloid-based chiral weak anion exchangers (WAX) and recently reported aminosulfonic acid-based chiral strong cation exchangers (SCX), which are conceptionally restricted to oppositely charged solutes, their individual chiral selector (SO) subunits have been fused in a combinatorial synthesis approach into single, now zwitterionic, chiral SO motifs. The corresponding zwitterionic ion-exchange-type chiral stationary phases (CSPs) in fact combined the applicability spectra of the parent chiral ion exchangers allowing for enantioseparations of chiral acids and amine-type solutes in liquid chromatography using polar organic mode with largely rivaling separation factors as compared to the parent WAX and SCX CSPs. Furthermore, the application spectrum could be remarkably expanded to various zwitterionic analytes such as alpha- and beta-amino acids and peptides. A set of structurally related yet different CSPs consisting of either a quinine or quinidine alkaloid moiety as anion-exchange subunit and various chiral or achiral amino acids as cation-exchange subunits enabled us to derive structure-enantioselectivity relationships, which clearly provided strong unequivocal evidence for synergistic effects of the two oppositely charged ion-exchange subunits being involved in molecular recognition of zwitterionic analytes by zwitterionic SOs driven by double ionic coordination.     

Thermoresponsive PEG‐Coated Nanotubes as Chiral Selectors of Amino Acids and Peptides

A series of nanotubes with a dense layer of short poly(ethylene glycol) (PEG) chains on the inner surface are prepared by means of a coassembly process using glycolipids and PEG derivatives. Dehydration of the PEG chains by heating increases the hydrophobicity of the nanotube channel and fluorescent‐dye‐labeled amino acids are extracted from bulk solution. Rehydration of the PEG chains by cooling results in back‐extraction of the amino acids into the bulk solution. Because of the supramolecular chirality of the nanotubes, amino acid enantiomers can be separated in the back‐extraction procedure, which is detectable with the naked eye as a change in fluorescence as the amino acids are released from the nanotubes. The efficiency and selectivity of the chiral separation are enhanced by tuning the chemical features and inner diameter of the nanotube channels. For example, compared with wide nanotube channels (8 nm), narrow nanotube channels (4 nm) provide more effective electrostatic attraction and hydrogen bond interaction environments for the transporting amino acids. Introduction of branched alkyl chains to the inner surface of the nanotubes enables chiral separation of peptides containing hydrophobic amino acids. The system described here provides a simple, quick, and on‐site chiral separation in biological and medical fields.     

Chiral analysis by electrospray ionization mass spectrometry/mass spectrometry. 2. Determination of enantiomeric excess of amino acids

The determination of enantiomeric excess (ee) of amino acids was achieved by investigating the collision-induced dissociation spectra of protonated trimers that were formed by electrospray ionization of amino acids in the presence of one of the following chiral selectors: L- or D-N-tert-butoxycarbonylphenylalanine, L- or D-N-tert-butoxycarbonylproline, and L- or D-N-tert-butoxycarbonyl-O-benzylserine. The protonated trimers were dissociated to form protonated dimers, and the observed dissociation efficiency r (i.e., the intensity ratio of protonated dimers to protonated trimers) for an enantiomeric mixture was found to be related to its ee value by the following equation: r = a + b/(c + ee), where a, b, and c were constants. A linear calibration plot was obtained by plotting r versus 1/(c + ee), where c was calculated with the MATLAB software, or by plotting 1/(r - r0) versus 1/ee, where r0 was the r value for the racemic mixture. The latter "two-reciprocal" method was more convenient for application. Another practical method for ee determination was the "three-point" method, whereby the ee of an unknown sample with a measured r value could be derived from the equation ee = 100?1/(rL - r0) - 1/(rD - r0)?/?2/(r - r0) - 1/(rL - r0) - 1/(rD - r0)?, with rL and rD being the r values for the enantiomerically pure L- and D-forms of the sample, respectively. A calibration plot was not required. The ee determination was achieved with acceptable precision even for the worst case of acceptable chiral recognition with a particular chiral selector, suggesting that the ee determination of all 19 common amino acids could be achieved by the present method. The ee of a histidine sample was determined both by the two-reciprocal method, giving an error of 0.2% ee (1.1% relative error) and consuming only approximately 5.3 nmol of sample, and by the three-point method, giving an error of 0.4% ee and consuming only approximately 2.3 nmol of sample. In the latter case, it took 27 min for the mass spectrometric measurements of the three calibration standards and an additional 9 min for the unknown sample. The direct ee determination of more than one amino acid in a mixture was also demonstrated in the study.     

Derivatization of chiral amino acids in supercritical carbon dioxide

A new method is proposed to perform the derivatization of chiral amino acids occurring in complex samples using supercritical carbon dioxide as both the reaction medium and the agent used to extract the obtained derivatives prior to accomplishing the subsequent enantiomeric chromatographic analysis. The derivatization step under supercritical conditions involves the esterification of the carboxyl group and the acylation of the amino group of the amino acids without using a catalyst. A Chirasil-L-Val capillary column enabled the separation of the D- and L-forms of the amino acids as their N(O)-pentafluoropropionyl 1-propyl esters. Relative standard deviation values obtained from the gas chromatographic analysis for the derivatized amino acids ranged from 5 to 15%.     

Validated quantitation of underivatized amino acids in human blood samples by volatile ion-pair reversed-phase liquid chromatography coupled to isotope dilution tandem mass spectrometry

Quantitation of amino acids in complex matrixes without derivatization is advantageous; however, difficulties exist in both the separation and the detection of those compounds. A validated method that is based on the use of volatile ion-pair liquid chromatography coupled to stable isotope dilution tandem mass spectrometry has been developed for the simple and accurate quantitation of underivatized amino acids in biological samples. Sufficient separation of 22 underivatized amino acids was achieved on a C18 column in 36 min using perfluoroheptanoic acid (PFHA) and trifluoroacetic acid (TFA) as mobile phase modifiers. The collisionally activated dissociation spectra of the amino acids were investigated and the transitions of [M + H]+ --> [M + H - 46]+, which are specific to alpha-amino acids, were used for the detection of most amino acids and their stable isotopes. The calibration curves were linear over the range of 0.10-100 microg/mL, and the detection limits were 0.03-20 pmol on column. The quantitative results by this method were compared with those by an established OPA-derivatization HPLC method in the assay of 8 human serum samples, and better recovery and precision data of this method were observed. The method was also applied to the neonatal screening for phenylketonuria (PKU) with dry blood spots, and the results were satisfactory. This is the first time that all proteinogenic amino acids have been quantified directly from biological extracts without any kind of derivaization. The technique shows potential for routine determination of amino acids and analogous compounds in complex matrixes.     

Chiral ionic liquid that functions as both solvent and chiral selector for the determination of enantiomeric compositions of pharmaceutical products

We have successfully synthesized both enantiomers of a novel chiral ionic liquid, (R)- and (S)-[(3-chloro-2-hydroxypropyl) trimethylammonium][bis((trifluoromethyl)sulfonyl)amide] ((R)- and (S)-[CHTA]+[Tf2N]-) in optically pure form by a simple ion exchange reaction from corresponding chloride salts that are commercially available. In addition to the ease of preparation, this chiral IL has relatively high thermal stability (up to 300 degrees C), is liquid at room temperature (glass transition temperature of -58.4 degrees C), and exhibits strong enantiomeric recognition. The high solubility power and strong enantiomeric recognition ability make it possible to use this chiral IL to solubilize an analyte and to induce diastereomeric interactions for the determination of enantiomeric purity. In fact, we have successfully developed a novel method based on the near-infrared technique with this chiral IL serving both as solvent and as a chiral selector for the determination of enantiomeric purity. Enantiomeric compositions of a variety of pharmaceutical products and amino acids with different shape, size, and functional groups can be sensitively (milligram concentration) and accurately (enantiomeric excess as low as 0.6%) determined by use of this method.     

Electrochemical detection of peptides

A general method of wide applicability for the determination of peptides is described. Peptides longer than dipeptides react in the classical biuret reaction with Cu(II) to yield electroactive Cu(II)-peptide complexes that can be oxidized to the corresponding Cu(III) complexes. This allows the sensitive electrochemical detection of peptides following their separation by reversed-phase liquid chromatography. The reaction chemistry, which is reversible, allows for the determination of peptides that lack an electroactive group or a primary amine. Selectivity for a model peptide is 10(3)-10(4) over nonelectroactive amino acids.     

Enantioseparation and stacking of Cyanobenz[f]isoindole-amino acids by reverse polarity capillary electrophoresis and sulfated beta-cyclodextrin

A capillary electrophoresis method with laser-induced fluorescence detection for the chiral separation of cyanobenz[f]isoindole (CBI) derivatives of amino acids was developed and optimized. The enantioseparations are accomplished with sulfated beta-CD (S-beta-CD) as chiral selector at low pH and reverse polarity. BGE conditions were optimized for CBI-serine and then applied to other CBI-amino acids. Baseline resolution of 13 CBI-amino acids was achieved using a single BGE formulation of 2 wt % S-beta-CD in 25 mM phosphate buffer at pH 2.00 and a voltage of -30 kV. pH is the most critical BGE parameter affecting resolution. At 2 wt % S-beta-CD, CBI-serine enantiomers are baseline-resolved at pH 2.00 but no resolution is obtained at pH 3.00. l-Glutamate, l-aspartate and d-serine are simultaneously quantified in the microdialysate of an arctic ground squirrel to illustrate the application to biological samples. Dilute solutions of the CBI-amino acids in water can be stacked by hydrodynamic injection with a 100-fold improvement in signal-to-noise ratio without loss of chiral resolution. The stacking is proposed to consist of field-amplified migration, pH-mediated stacking, and sweeping by S-beta-CD. The limit of detections for CBI-dl-serine and CBI-dl-glutamate are determined as 0.20 and 0.30 nM, respectively. The stacking method was not applicable to the high ionic strength microdialysates.     

Distinguishing and quantifying peptides and proteins containing D-amino acids by tandem mass spectrometry

Tandem mass spectrometry (MS/MS) utilizing both electron capture dissociation (ECD) and collisionally activated dissociation (CAD) was used to develop a qualitative and quantitative analytical method for chiral analysis of individual amino acid residues in polypeptides. ECD produced a more distinct chiral recognition than CAD, which is attributed to the smaller degree of vibrational excitation in ECD. Several peptide and protein model systems were used in this study, including the smallest known protein, tryptophan cage, a lactoferrin peptide, and the biologically relevant opioid peptide, dermorphin. An adaptation of the kinetic method was used to quantify the degree of separation between fragmentation patterns of stereoisomeric peptides as a function of fragment ion abundances. The obtained calibration scale for relative abundances of d-amino acids in diastereomeric peptide mixtures was accurate to 1% for ECD and to 3-5% for CAD. It was found that separation and quantification of stereoisomers could be advantageously performed by nanoflow reversed-phase liquid chromatography, with the objective of on-line MS/MS limited to stereoisomer identification. This technique shows promise for the analysis of chiral substitution in peptides and proteins, broadening the application area for tandem mass spectrometry.     

Dioxin-binding pentapeptide for use in a high-sensitivity on-bead detection assay

The purpose of this study is to develop a dioxin detection method using a short peptide alternative to an immunoantibody. A full peptide library consisting of 2.5 million possible amino acid combinations was constructed by a solid-phase split synthesis approach using 19 natural amino acids. The peptide beads were subjected to a competitive binding assay between 2,3,7-trichlorodibenzo-p-dioxin and N-NBD-3-(3',4'-dichlorophenoxy)-1-propylamine (NBD-DCPPA) in a buffer containing 20% 1,4-dioxane. Two almost identical pentapeptides, FLDQI and FLDQV, that could bind dioxin were screened from the combinatorial library. NBD-DCPPA and the peptide synthesized on resin beads could be utilized to determine dioxin concentrations. The fluorescence intensity of the beads was measured using fluorescence microscopy to make a calibration curve for the dioxin concentrations. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TeCDD) could also detected in the presence of 30% 1,4-dioxane. To optimize the peptide sequence, a one-amino acid-substituted library was prepared using amino acids including nonnatural amino acids. The internal amino acids, LDQ, could not be substituted by any other amino acids. This result indicates that these three side chains are essential to recognize dioxins. The peptide C terminus substituted by phenylglycine showed a 10 times lower detection limit of 2,3,7,8-TeCDD of 150 pM (50 pg/mL) than the original sequence FLDQV. The cross reactivity of the dioxin binding peptides including the secondary derivatives was investigated. Some polycyclic aromatic hydrocarbons bound to the peptide beads, but nonchlorinated dibenzo-p-dioxin and PCB did not. From these results, we demonstrate the potential of short peptides as a practical sensor material targeting low molecular weight compounds such as dioxin.     

Ion-pair RP-HPLC determination of sugars, amino sugars, and uronic acids after derivatization with p-aminobenzoic acid

A new, selective, and sensitive ion-pair RP-HPLC method for the simultaneous determination of three classes of natural organic compounds, i.e., carbohydrates, amino sugars, and uronic acids, in environmental samples is presented. p-Aminobenzoic acid is used for precolumn derivatization of the analytes, enabling fluorescence (lambda(ex) 313 nm, lambda(em) 358 nm) or photometric detection (303 nm). The dependence of the derivatization yield on the reaction conditions is examined. Derivatives of lactose, galactose, glucose, mannose, xylose, arabinose, galacturonic acid, glucuronic acid, N-acetylglucosamine, and glycerinealdehyde were separated on a RP-C18 column with hydrophilic end capping within 35 min, applying TBAHSO4 as the ion-pair reagent. The concentration detection limits range between 20 and 30 microg L(-1) ((1-2) x 10(-7) M) for fluorescence detection and between 30 and 75 microg L(-1) for UV detection. A good linearity is achieved in the concentration range from 50 microg L(-1) to 100 mg L(-1) (r2 > 0.99). The described method has been applied for the determination of mono-/disaccharides, uronic acids, and amino sugars in soil solutions and in landfill leachates.     

多肽和氨基酸纳滤膜分离中的膜污染及防治研究进展

综述了近期有关多肽和氨基酸的纳滤膜分离过程中的膜污染及防治的研究进展。膜表面浓差极化层或凝胶层的形成以及多肽和氨基酸与膜的疏水或静电吸附是导致该过程膜污染的主要原因，调节溶液的物化条件，改善膜材质的性能和提高亲水性是当前膜污染防治的主要途径。     

Regenerable and simultaneous surface plasmon resonance detection of aβ(1-40) and aβ(1-42) peptides in cerebrospinal fluids with signal amplification by streptavidin conjugated to an N-terminus-specific antibody

A major constituent in the deposit of the brain in a patient with Alzheimer's disease (AD) is the aggregates/fibrils of amyloid-β (Aβ) peptides containing 39-43 amino acids. The total Aβ levels and the concentration ratio between the most abundant Aβ(1-40) peptide and the more aggregation-prone Aβ(1-42) in body fluids (e.g., cerebrospinal fluid or CSF) have been suggested as possible criteria for early diagnosis of AD. By immobilizing capture antibodies specific to the two peptides in separate fluidic channels, surface plasmon resonance (SPR) has been used to quantify Aβ(1-40) and Aβ(1-42) present in CSF samples collected from AD patients and healthy donors. With signal amplification by streptavidin conjugated to an antibody that is selective to the common N-terminus of the Aβ peptides, concentrations as low as 20 pM can be readily measured. The range of Aβ peptide concentrations measurable by this method spans 4 orders of magnitude. The ability of regenerating the sensor surface for repeated measurements not only improves the reproducibility but also enhances the sample throughput. Our data reveal that the ratio of Aβ(1-40) concentration versus Aβ(1-42) concentration in CSF samples from AD patients is almost twice as high as that from healthy persons. In contrast to the commonly used enzyme-linked immunosorbent assay (ELISA), SPR obviates the need of a more expensive and less stable enzyme conjugate and the use of carcinogenic substrate for the signal detection and allows the binding events to be monitored in real time.     

Chiral melamine derivatives: design, synthesis, and application to mass spectrometry-based chiral analysis

A novel class of chiral melamine derivatives has been designed and synthesized. The ability of these compounds to perform chiral recognition toward 19 natural chiral alpha-amino acids has been investigated by electrospray ionization tandem mass spectrometry for the first time. The enantioselectivities of these new chiral selectors are encouraging. To elucidate some mechanism and regularity in the chiral recognition process using chiral melamine derivatives as chiral selectors, the effect of different noncovalent interactions caused by various chiral or achiral moieties in melamine derivatives on the chiral recognition in the gas phase has been studied at the same time. The result shows that electrostatic, hydrogen bond, pi-pi stacking, and steric interaction between selector and analyte play important roles in the association and enantioselective recognition of amino acids with the chiral melamine derivatives as chiral selectors. Enantiodiscrimination for analytes with different structures and properties could be improved by modifying substituents in melamine derivatives on purpose.     

Enantioselective recognition of a pair of novel receptors for amino acid zwitterions

A pair of artificial enantiotopic receptors 1a, 1b composed of (S,S) or (R,R) chiral bicyclic guanidinium, azacrown ether and (t-butyldiphenylsilyloxy)methyl group for amino acid zwitterions have been synthesized. The liquid–liquid competition extraction experiments and ¹H NMR studies indicate that the receptor 1a with (S,S) configuration and 1b with (R,R) configuration selectively recognize - and -aromatic amino acids, respectively.     

Direct high-performance liquid chromatographic separation of peptide enantiomers: study on chiral recognition by systematic evaluation of the influence of structural features of the chiral selectors on enantioselectivity

All-R/all-S enantiomers of oligoalanines (Ala(n), n = 1-10) with N-terminal protection group have been separated by HPLC on chiral stationary phases based on various cinchona alkaloid selectors. Structure-enantioselectivity relationships derived by extensive selector structure optimization provided insights into binding mechanisms and chiral recognition. Their interpretation was supported by X-ray crystal structures of amino acid and dipeptide, respectively, in complex with chiral selector. Optimized selectors have bulky elements representing steric barriers and deep binding pockets that afforded very high enantioselectivities; e.g., for the all-R and all-S enantiomers of N-(3,5-dinitrobenzoyl)alanylalanine, an alpha-value of 20.0 (corresponding to deltadeltaG of -7.43 kJ/mol) was obtained with a chiral stationary phase based on 6'-(neopentoxy)-9-O-tert-butylcarbamoylcinchonidine. Further, a chiral stationary phase based on 1,4-bis(9-O-quinidinyl)phthalazine was able to distinguish between the all-R and all-S enantiomers of hepta- to decaalanine peptides with enantioselectivity values between 1.8 and 1.9, corresponding to deltadeltaG of -1.46 and -1.59 kJ/mol, respectively.