D-Amino acids in aged proteins: analysis and biological relevance

Homochirality is essential for life. L-Amino acids are exclusively used as substrates for the polymerization and formation of peptides and proteins in living systems. However, d-amino acids, which are enantiomers of L-amino acids, were recently detected in various living organisms in the form of free D-amino acids and D-amino acid residues in peptides and proteins. In particular, D-aspartyl (Asp) residues have been detected in various proteins from diverse tissues of elderly individuals. Here, we describe three important aspects of our research: (i) a method for detecting D-β-Asp at specific sites in particular proteins, (ii) a likely spontaneous mechanism by which Asp residues in proteins invert and isomerize to the D-β-form with age under physiological conditions, (iii) a discussion of factors that favor such a reaction.     

Breaking the mirror: l-Amino acid deaminase,a novel stereoselective biocatalyst

Enantiomerically pure amino acids are of increasing interest for the fine chemical, agrochemicals and pharmaceutical industries. During past years l-amino acids have been produced from deracemization of dl-solution employing the stereoselective flavoenzyme d-amino acid oxidase. On the other hand, the isolation of corresponding d-isomer was hampered by the scarce availability of a suitable l-amino acid oxidase activity. On this side, l-amino acid deaminase (LAAD), only present in the Proteus bacteria, represents a suitable alternative. This FAD-containing enzyme catalyzes the deamination of l-amino acids to the corresponding α-keto acids and ammonia, with no hydrogen peroxide production (a potentially dangerous oxidizing species) since the electrons of the reduced cofactor are transferred to a membrane-bound cytochrome. Very recently the structure of LAAD has been solved: in addition to a FAD-binding domain and to a substrate-binding domain, it also possesses an N-terminal putative transmembrane α-helix (residues 8–27, not present in the crystallized protein variant) and a small α + β subdomain (50–67 amino acids long, named “insertion module”) strictly interconnected to the substrate binding domain. Structural comparison showed that LAAD resembles the structure of several soluble amino acid oxidases, such as l-proline dehydrogenase, glycine oxidase or sarcosine oxidase, while only a limited structural similarity with d- or l-amino acid oxidase is apparent. In this review, we present an overview of the structural and biochemical properties of known LAADs and describe the advances that have been made in their biotechnological application also taking advantage from improved variants generated by protein engineering studies.     

Production of aromatic d-amino acids from α-keto acids and ammonia by coupling of four enzyme reactions

A multi-enzyme system composed of glutamate racemase, thermostable d-amino acid aminotransferase, glutamate dehydrogenase and formate dehydrogenase was employed for the production of aromatic d-amino acids, d-phenylalanine and d-tyrosine, from the corresponding α-keto acids, phenylpyruvate and hydroxyphenylpyruvate, respectively. The optimal concentration of ammonium formate for the production of these d-amino acids was found in the range of 0.25–1.0 M. The optimal concentration of α-keto acid was determined to be 50 mM, above which the productivity greatly decreased. To keep the concentration of α-keto acid around this concentration, α-keto acid was intermittently fed into the multi-enzyme system during the production period. By running the multi-enzyme system for 35 h, 48 g l⁻¹ of d-phenylalanine and 60 g l⁻¹ of d-tyrosine were produced with 100% of optical purity from the equimolar amounts of phenylpyruvate and hydroxyphenylpyruvate, respectively. The production levels of both aromatic d-amino acids were demonstrated to be dependent on the stability of glutamate racemase.     

Construction of d-amino acid biosensor based on d-amino acid oxidase immobilized onto poly (indole-5-carboxylic acid)/zinc sulfide nanoparticles hybrid film

d-Amino acid oxidase (DAAO) purified from goat kidney was immobilized covalently via N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry onto poly indole 5-carboxylic acid (Pin5-COOH)/zinc sulfide nanoparticles (ZnSNPs) hybrid film electrodeposited on surface of an Au electrode. A highly sensitive d-amino acid biosensor was constructed using this enzyme electrode as working electrode, Ag/AgCl as reference electrode, and Pt wire as auxiliary electrode connected through potentiostat. The biosensor showed optimum response within 3 s at pH 7.5 and 35 °C, when polarized at 0.15 V vs. Ag/AgCl. There was a linear relationship between biosensor response (mA) and d-alanine concentration in the range 0.001–2.0 mM. The sensitivity of the biosensor was 58.85 μA cm^?2 mM^?1 with a detection limit of 0.001 mM (S/N = 3). The enzyme electrode was used 120 times over a period of 2 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective layer of poly indole-5-carboxylic acid film. The biosensor was evaluated and employed for measurement of d-amino acid level in fruits and vegetables.     

Comparison of the activation energy barrier for succinimide formation from α- and β-aspartic acid residues obtained from density functional theory calculations

The l-α-Asp residues in peptides or proteins are prone to undergo nonenzymatic reactions to form l-β-Asp, d-α-Asp, and d-β-Asp residues via a succinimide five-membered ring intermediate. From these three types of isomerized aspartic acid residues, particularly d-β-Asp has been widely detected in aging tissue. In this study, we computationally investigated the cyclization of α- and β-Asp residues to form succinimide with dihydrogen phosphate ion as a catalyst (H₂PO₄⁻). We performed the study using B3LYP/6-31 + G(d,p) density functional theory calculations. The comparison of the activation barriers of both residues is discussed. All the calculations were performed using model compounds in which an α/β-Asp-Gly sequence is capped with acetyl and methylamino groups on the N- and C-termini, respectively. Moreover, H₂PO₄⁻ catalyzes all the steps of the succinimide formation (cyclization-dehydration) acting as a proton-relay mediator. The calculated activation energy barriers for succinimide formation of α- and β-Asp residues are 26.9 and 26.0 kcal mol^− 1, respectively. Although it was experimentally confirmed that β-Asp has higher stability than α-Asp, there was no clear difference between the activation barriers. Therefore, the higher stability of β-Asp residue than α-Asp residue may be caused by an entropic effect associated with the succinimide formation.     

Antimicrobial peptides containing unnatural amino acid exhibit potent bactericidal activity against ESKAPE pathogens

A series of 36 synthetic antimicrobial peptides containing unnatural amino acids were screened to determine their effectiveness to treat Enterococcus faecium, Staphylococcus aureus, Klebsiella pnemoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE) pathogens, which are known to commonly infect chronic wounds. The primary amino acid sequences of these peptides incorporate either three or six dipeptide units consisting of the unnatural amino acids Tetrahydroisoquinolinecarboxylic acid (Tic) and Octahydroindolecarboxylic acid (Oic). The Tic-Oic dipeptide units are separated by SPACER amino acids with specific physicochemical properties that control how these peptides interact with bacterial cell membranes of different chemical compositions. These peptides exhibited minimum inhibitory concentrations (MIC) against these pathogens in the range from >100 to 6.25 μg/mL. The observed diversity of MIC values for these peptides against the various bacterial strains are consistent with our hypothesis that the complementarity of the physicochemical properties of the peptide and the lipid of the bacteria’s cell membrane determines the resulting antibacterial activity of the peptide.     

Mammalian ALOX15 orthologs exhibit pronounced dual positional specificity with docosahexaenoic acid

Mammalian lipoxygenases (LOX) have been implicated in cell differentiation and in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. Although the reaction specificity of mammalian LOX with n − 6 fatty acids (linoleic acid, arachidonic acid) has been explored in detail little information is currently available on the product patterns formed from n − 3 polyenoic fatty acids, which are of particular nutritional importance and serve as substrate for the biosynthesis of pro-resolving inflammatory mediators such as resolvins and maresins. Here we expressed the ALOX15 orthologs of eight different mammalian species as well as human ALOX12 and ALOX15B as recombinant his-tag fusion proteins and characterized their reaction specificity with the most abundantly occurring polyunsaturated fatty acids (PUFAs) including 5,8,11,14,17-eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-docosahexaenoic acid (DHA). We found that the LOX isoforms tested accept these fatty acids as suitable substrates and oxygenate them with variable positional specificity to the corresponding n − 6 and n − 9 hydroperoxy derivatives. Surprisingly, human ALOX15 as well as the corresponding orthologs of chimpanzee and orangutan, which oxygenates arachidonic acid mainly to 15S-H(p)ETE, exhibit a pronounced dual reaction specificity with DHA forming similar amounts of 14- and 17-H(p)DHA. Moreover, ALOX15 orthologs prefer DHA and EPA over AA when equimolar concentrations of n − 3 and n − 6 PUFA were supplied simultaneously. Taken together, these data indicate that the reaction specificity of mammalian LOX isoforms is variable and strongly depends on the chemistry of fatty acid substrates. Most mammalian ALOX15 orthologs exhibit dual positional specificity with highly unsaturated n − 3 polyunsaturated fatty acids.     

Purification and identification of an angiotensin I-converting enzyme inhibitory peptide from fermented soybean extract

Angiotensin I-converting enzyme (ACE), a dipeptidyl carboxypeptidase, plays an important physiological role in regulating blood pressure. ACE-inhibitory peptides derived from food proteins have potential pharmaceutical and human health uses. In this study, we prepared a fermented soybean extract (FSE) through a rapid fermentation at an elevated temperature to accelerate proteolytic hydrolysis and described purification procedures to discover potent ACE-inhibitory peptides from FSE. After 3 days of aging, FSE exhibited ACE-inhibitory activity with an IC₅₀ value of 1.46 mg/mL. Purification of novel ACE-inhibitory peptides was carried out using ultrafiltration and consecutive chromatographic methods. A novel ACE-inhibitory peptide, with 66-fold increase in ACE-inhibitory activity compared to that of FSE, was isolated from FSE through a five-step purification procedure. The amino acid sequence of the purified ACE-inhibitory peptides was determined to be Leu-Val-Gln-Gly-Ser by Edman degradation method, and its IC₅₀ value was 22 μg/mL (43.7 μM).     

Cloning,expression and characterization of d-aminoacylase from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173

d-Aminoacylase catalyzes the conversion of N-acyl-d-amino acids to d-amino acids and fatty acids. The aim of this study was to identify the d-aminoacylase gene from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173 and investigate the biochemical characterization of the enzyme. A previously uncharacterized d-aminoacylase gene (ADdan) from this organism was cloned and sequenced. The open reading frame (ORF) of ADdan was 1467 bp in size encoding a 488-amino acid polypeptide. ADdan, with a high amino acid similarity to N-acyl-d-aspartate amidohydrolase from Alcaligenes A6, showed relatively low sequence similarities to other characterized d-aminoacylases. The recombinant ADdan protein was expressed in Escherichia coli BL21 (DE3) using pET-28a with a T7 promoter. The enzyme was purified in a single chromatographic step using nickel affinity gel column. The molecular mass of the expressed protein, calculated by SDS–PAGE, was about 52 kDa. The purified ADdan showed optimal activity at pH 8.0 and 50 °C, and was stable at pH 6.0–8.0 and up to 45 °C. Its activity was inhibited by Cu²⁺, Fe²⁺, Ca²⁺, Mn²⁺, Ni²⁺, Zn²⁺ and Hg²⁺, whereas Mg²⁺ had no significant influence on this recombinant d-aminoacylase. This is the first report on the characterization of d-aminoacylase with activity towards both N-acyl derivatives of neutral d-amino acids and N-acyl-d-aspartate. The characteristics of ADdan could prove to be of interest in industrial production of d-amino acids.     

Antimicrobial properties of two novel peptides derived from Theobroma cacao osmotin

The osmotin proteins of several plants display antifungal activity, which can play an important role in plant defense against diseases. Thus, this protein can be useful as a source for biotechnological strategies aiming to combat fungal diseases. In this work, we analyzed the antifungal activity of a cacao osmotin-like protein (TcOsm1) and of two osmotin-derived synthetic peptides with antimicrobial features, differing by five amino acids residues at the N-terminus. Antimicrobial tests showed that TcOsm1 expressed in Escherichia coli inhibits the growth of Moniliophthora perniciosa mycelium and Pichia pastoris X-33 in vitro. The TcOsm1-derived peptides, named Osm-pepA (H-RRLDRGGVWNLNVNPGTTGARVWARTK-NH₂), located at R23-K49, and Osm-pepB (H-GGVWNLNVNPGTTGARVWARTK-NH₂), located at G28-K49, inhibited growth of yeasts (Saccharomyces cerevisiae S288C and Pichia pastoris X-33) and spore germination of the phytopathogenic fungi Fusarium f. sp. glycines and Colletotrichum gossypi. Osm-pepA was more efficient than Osm-pepB for S. cerevisiae (MIC = 40 μM and MIC = 127 μM, respectively), as well as for P. pastoris (MIC = 20 μM and MIC = 127 μM, respectively). Furthermore, the peptides presented a biphasic performance, promoting S. cerevisiae growth in doses around 5 μM and inhibiting it at higher doses. The structural model for these peptides showed that the five amino acids residues, RRLDR at Osm-pepA N-terminus, significantly affect the tertiary structure, indicating that this structure is important for the peptide antimicrobial potency. This is the first report of development of antimicrobial peptides from T. cacao. Taken together, the results indicate that the cacao osmotin and its derived peptides, herein studied, are good candidates for developing biotechnological tools aiming to control phytopathogenic fungi.     

Complete amino acid analysis of peptides and proteins after hydrolysis by a mixture of Sepharose-bound peptidases

Incubation with a mixture of Sepharose-bound peptidases was shown to result in the quantitative release of amino acids from certain peptides and S-aminoethylated proteins. Subtraction of the low background values of amino acids generated by the enzymes enables amino acid ratios of corticotrophin-(1-24)-tetracosapeptide to be determined with a standard deviation on repeat digestions of 3-5%. Good values were obtained for amino acids that are completely or partially destroyed on acid hydrolysis, i.e. tryptophan, tyrosine, serine, asparagine and glutamine. Experiments with peptides containing d-amino acids showed that the enzyme mixture is stereospecific and could therefore be used to detect the presence of d-residues in peptides. The enzyme mixture completely hydrolyses peptide fragments obtained after Edman degradation and should therefore be useful for determining sequences of peptides containing acid-labile amino acid residues. The activities of the bound enzymes were unaltered over a period of 7 months and they provide a simple, reproducible procedure for the quantitative determination of amino acids in peptides and proteins containing l-amino acids.     

Toward the inhibitory effect of acetylsalicylic acid on tyrosinase: Integrating kinetics studies and computational simulations

Acetylsalicylic acid (ASA), generally well known as aspirin, has various biomedical functions. In this study, we revealed that ASA reversibly inhibits tyrosinase (EC 1.14.18.1) in a mixed-type manner with a K_i = 11.778 ± 2.01 mM. Time-interval kinetics showed that the inhibition followed first-order reaction kinetics. Measurements of ANS-binding fluorescence showed that ASA did not induce significant detectable changes in the hydrophobic surface of tyrosinase. For further insight, we performed molecular dynamics simulations to predict the key interactions between tyrosinase and ASA and found that the acetate and carboxylic acid groups of ASA play a critical role in binding to several residues (HIS61, HIS85, HIS94, HIS259, HIS263, and ALA286) on tyrosinase that are thought to be pivotal for docking. Our study suggested that ASA could be a useful depigmentation agent due to the structural functions of the acetic and carboxyl groups on tyrosinase.     

Characterization of d-amino acid aminotransferase from Lactobacillus salivarius

We searched a UniProt database of lactic acid bacteria in an effort to identify d-amino acid metabolizing enzymes other than alanine racemase. We found a d-amino acid aminotransferase (d-AAT) homologous gene (UniProt ID: Q1WRM6) in the genome of Lactobacillus salivarius. The gene was then expressed in Escherichia coli, and its product exhibited transaminase activity between d-alanine and α-ketoglutarate. This is the first characterization of a d-AAT from a lactic acid bacterium. L. salivarius d-AAT is a homodimer that uses pyridoxal-5′-phosphate (PLP) as a cofactor; it contains 0.91 molecules of PLP per subunit. Maximum activity was seen at a temperature of 60 °C and a pH of 6.0. However, the enzyme lost no activity when incubated for 30 min at 30 °C and pH 5.5 to 9.5, and retained half its activity when incubated at pH 4.5 or 11.0 under the same conditions. Double reciprocal plots of the initial velocity and d-alanine concentrations in the presence of several fixed concentrations of α-ketoglutarate gave a series of parallel lines, which is consistent with a Ping-Pong mechanism. The K_m values for d-alanine and α-ketoglutarate were 1.05 and 3.78 mM, respectively. With this enzyme, d-allo-isoleucine exhibited greater relative activity than d-alanine as the amino donor, while α-ketobutylate, glyoxylate and indole-3-pyruvate were all more preferable amino acceptors than α-ketoglutarate. The substrate specificity of L. salivarius d-AAT thus differs greatly from those of the other d-AATs so far reported.     

Global survey of the omega-3 fatty acids,docosahexaenoic acid and eicosapentaenoic acid in the blood stream of healthy adults

Studies reporting blood levels of the omega-3 polyunsaturated fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were systematically identified in order to create a global map identifying countries and regions with different blood levels. Included studies were those of healthy adults, published in 1980 or later. A total of 298 studies met all inclusion criteria. Studies reported fatty acids in various blood fractions including plasma total lipids (33%), plasma phospholipid (32%), erythrocytes (32%) and whole blood (3.0%). Fatty acid data from each blood fraction were converted to relative weight percentages (wt.%) and then assigned to one of four discrete ranges (high, moderate, low, very low) corresponding to wt.% EPA + DHA in erythrocyte equivalents. Regions with high EPA + DHA blood levels (> 8%) included the Sea of Japan, Scandinavia, and areas with indigenous populations or populations not fully adapted to Westernized food habits. Very low blood levels (≤ 4%) were observed in North America, Central and South America, Europe, the Middle East, Southeast Asia, and Africa. The present review reveals considerable variability in blood levels of EPA + DHA and the very low to low range of blood EPA + DHA for most of the world may increase global risk for chronic disease.     

Determination of stereochemistry stability coefficients of amino acid side-chains in an amphipathic alpha-helix.

We describe here a systematic study to determine the effect on secondary structure of d-amino acid substitutions in the nonpolar face of an amphipathic alpha-helical peptide. The helix-destabilizing ability of 19 d-amino acid residues in an amphipathic alpha-helical model peptide was evaluated by reversed-phase HPLC and CD spectroscopy. l-Amino acid and d-amino acid residues show a wide range of helix-destabilizing effects relative to Gly, as evidenced in melting temperatures (DeltaTm) ranging from -8.5 degrees C to 30.5 degrees C for the l-amino acids and -9.5 degrees C to 9.0 degrees C for the d-amino acids. Helix stereochemistry stability coefficients defined as the difference in Tm values for the l- and d-amino acid substitutions [(DeltaTm' = TmL and TmD)] ranging from 1 degrees C to 34.5 degrees C. HPLC retention times [DeltatR(XL-XD)] also had values ranging from -0.52 to 7.31 min at pH 7.0. The helix-destabilizing ability of a specific d-amino acid is highly dependent on its side-chain, with no clear relationship to the helical propensity of its corresponding l-enantiomers. In both CD and reversed-phase HPLC studies, d-amino acids with beta-branched side-chains destabilize alpha-helical structure to the greatest extent. A series of helix stability coefficients was subsequently determined, which should prove valuable both for protein structure-activity studies and de novo design of novel biologically active peptides.     

Mass spectral characterization of organophosphate-labeled,tyrosine-containing peptides: Characteristic mass fragments and a new binding motif for organophosphates

We have identified organophosphorus agent (OP)-tyrosine adducts on 12 different proteins labeled with six different OP. Labeling was achieved by treating pure proteins with up to 40-fold molar excess of OP at pH 8–8.6. OP-treated proteins were digested with trypsin, and peptides were separated by HPLC. Fragmentation patterns for 100 OP-peptides labeled on tyrosine were determined in the mass spectrometer. The goals of the present work were (1) to determine the common features of the OP-reactive tyrosines, and (2) to describe non-sequence MSMS fragments characteristic of OP-tyrosine peptides. Characteristic ions at 272 and 244 amu for tyrosine-OP immonium ions were nearly always present in the MSMS spectrum of peptides labeled on tyrosine by chlorpyrifos-oxon. Characteristic fragments also appeared from the parent ions that had been labeled with diisopropylfluorophosphate (216 amu), sarin (214 amu), soman (214 amu) or FP-biotin (227, 312, 329, 691 and 708 amu). In contrast to OP-reactive serines, which lie in the consensus sequence GXSXG, the OP-reactive tyrosines have no consensus sequence. Their common feature is the presence of nearby positively charged residues that activate the phenolic hydroxyl group. The significance of these findings is the recognition of a new binding motif for OP to proteins that have no active site serine. Modified peptides are difficult to find when the OP bears no radiolabel and no tag. The characteristic MSMS fragment ions are valuable because they are identifiers for OP-tyrosine, independent of the peptide.     

The role of tryptophans on the cellular uptake and membrane interaction of arginine-rich cell penetrating peptides

Cell-penetrating peptides (CPP) are able to efficiently transport cargos across cell membranes without being cytotoxic to cells, thus present a great potential in drug delivery and diagnosis. While the role of cationic residues in CPPs has been well studied, that of Trp is still not clear. Herein 7 peptide analogs of RW9 (RRWWRRWRR, an efficient CPP) were synthesized in which Trp were systematically replaced by Phe residues. Quantification of cellular uptake reveals that substitution of Trp by Phe strongly reduces the internalization of all peptides despite the fact that they strongly accumulate in the cell membrane. Cellular internalization and biophysical studies show that not only the number of Trp residues but also their positioning in the helix and the size of the hydrophobic face they form are important for their internalization efficacy, the highest uptake occurring for the analog with 3 Trp residues. Using CD and ATR-FTIR spectroscopy we observe that all peptides became structured in contact with lipids, mainly in α-helix. Intrinsic tryptophan fluorescence studies indicate that all peptides partition in the membrane in about the same manner (Kp ~ 10⁵) and that they are located just below the lipid headgroups (~ 10 Å) with slightly different insertion depths for the different analogs. Plasmon Waveguide Resonance studies reveal a direct correlation between the number of Trp residues and the reversibility of the interaction following membrane washing. Thus a more interfacial location of the CPP renders the interaction with the membrane more adjustable and transitory enhancing its internalization ability.     

Molecular cloning and characterization of a recombinant Bombyx mori tyramine-β-hydroxylase in a silkworm cell line using a baculovirus expression vector system

Octopamine (OA) and tyramine (TA) are biogenic amines that act as neurotransmitters, neurohormones, and neuromodulators in the invertebrate nervous system. Tyramine-β-hydroxylase (TβH) catalyzes the biosynthesis of OA from TA. In this study, cDNA encoding Bombyx mori TβH (BmTβH) was cloned from the brain of the silkworm B. mori. The BmTβH mRNA comprised 2204 nucleotide residues and contained an open reading frame encoding 592 amino acids. The deduced amino acid sequence shared homology to several proteins belonging to the insect TβH family. Functional expression of the cloned cDNA was obtained using a B. mori baculovirus expression vector system. Western blot analysis revealed an immunoreactive band with a molecular mass of ~ 67.4 kDa. Reverse-phase high-performance liquid chromatography (HPLC) was used to identify the products formed during incubation of the enzyme reaction mixture. The optimum pH and temperature for the conversion of TA to OA were 7.5 and 25 °C, respectively. During incubation, the reaction was linear for the first 30 min at 25 °C and pH 7.5. Inhibitory experiments carried out with various concentrations of an inhibitor showed that this method can be used for screening of BmTβH inhibitors.     

Novel endosomolytic peptides for enhancing gene delivery in nanoparticles

Trapping in the endosomes is currently believed to represent the main barrier for transfection. Peptides, which allow endosomal escape have been demonstrated to overcome this barrier, similarly to the entry of viruses. However, the design principles of such endosomolytic peptides remain unclear. We characterized three analogs derived from membrane disrupting antimicrobial peptides (AMP), viz. LL-37, melittin, and bombolitin V, with glutamic acid substituting for all basic residues. These analogs are pH-sensitive and cause negligible membrane permeabilization and insignificant cytotoxicity at pH 7.4. However, at pH 5.0, prevailing in endosomes, membrane binding and hemolysis of human erythrocytes become evident. We first condensed the emerald green fluorescent protein (emGFP) containing plasmid by protamine, yielding 115 nm diameter soluble nanoplexes. For coating of the nanoplex surface with a lipid bilayer we introduced a hydrophobic tether, stearyl-octa-arginine (SR8). The indicated peptides were dissolved in methanol and combined with lipid mixtures in chloroform, followed by drying at RT under a nitrogen flow. The dry residues were hydrated with nanoplexes in Hepes, pH 7.4 yielding after a 30 min incubation at RT,rather monodisperse nanoparticles having an average diameter of 150–300 nm, measured by DLS and cryo-TEM. Studies with cell cultures showed the above peptides to yield expression levels comparable to those obtained using Lipofectamine 2000. However, unlike the polydisperse aggregates formed upon mixing Lipofectamine 2000 and plasmid, the procedure described yields soluble, and reasonably monodisperse nanoparticles, which can be expected to be suitable for gene delivery in vivo, using intravenous injection.     

Urinary amino acid analysis: A comparison of iTRAQ®–LC–MS/MS,GC–MS,and amino acid analyzer

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) of propyl chloroformate and iTRAQ^® derivatized amino acids, respectively, to conventional amino acid analysis. The GC–MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC–MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ^® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC–MS, and iTRAQ^®–LC–MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27 ± 5.22, 21.18 ± 10.94, and 18.34 ± 14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39 ± 5.35, 6.23 ± 3.84, and 35.37 ± 29.42. Both GC–MS and iTRAQ^®–LC–MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.