Affinity Purification of Methyllysine Proteome by Site-Specific Covalent Conjugation.

A basic but critical step in targeted proteomics by mass spectrometry is the separation of the targeted proteins from the complex mixture of the whole proteome by affinity purification. The bait protein is usually immobilized on the surface of a solid support to enable affinity-based purification of the targeted proteome. Here, we developed a site-specific covalent immobilization of the bait protein through affinity-guided covalent coupling (AGCC) of a single cysteine residue of an SH2 domain (utilized as an affinity tag for the protein target) with an engineered ligand peptide. Site-specific covalent immobilization of a methyllysine-binding protein HP1ß chromodomain on the agarose resin was used to purify the methyllysine proteome from the whole-protein mixture. This new bait immobilization led to a notably low background in the affinity purification step, markedly outperforming the conventional (His)6 tag-nickel nitrilotriacetic acid (Ni-NTA) immobilization method. Subsequent analysis of the purified proteome identified 275 lysine methylated sites and 184 methylated proteins from 332 HP1ß CD-binding proteins, including 30 novel methylated proteins. This work demonstrates that a robust site-specific covalent protein immobilization method is well-suited for proteomic analysis of low-abundance proteins. This method also enables the identification of new methylated proteins and methylation sites in the methyllysine proteome.

l-Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene.

We have recently developed a Corynebacterium glutamicum strain that generates NADPH via the glycolytic pathway by replacing endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GapA) with a nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans. Strain RE2, a suppressor mutant spontaneously isolated for its improved growth on glucose from the engineered strain, was proven to be a high-potential host for l-lysine production (Takeno et al., 2010). In this study, the suppressor mutation was identified to be a point mutation in rho encoding the transcription termination factor Rho. Strain RE2 still showed retarded growth despite the mutation rho696. Our strategy for reconciling improved growth with a high level of l-lysine production was to use GapA together with GapN only in the early growth phase, and subsequently shift this combination-type glycolysis to one that depends only on GapN in the rest of the growth phase. To achieve this, we expressed gapA under the myo-inositol-inducible promoter of iolT1 encoding a myo-inositol transporter in strain RE2. The resulting strain RE2A(iol) was engineered into an l-lysine producer by introduction of a plasmid carrying the desensitized lysC, followed by examination for culture conditions with myo-inositol supplementation. We found that as a higher concentration of myo-inositol was added to the seed culture, the following fermentation period became shorter while maintaining a high level of l-lysine production. This finally reached a fermentation period comparable to that of the control GapA strain, and yielded a 1.5-fold higher production rate compared with strain RE2. The transcript level of gapA, as well as the GapA activity, in the early growth phase increased in proportion to the myo-inositol concentration and then fell to low levels in the subsequent growth phase, indicating that improved growth was a result of increased GapA activity, especially in the early growth phase. Moreover, blockade of the pentose phosphate pathway through a defect in glucose 6-phosphate dehydrogenase did not significantly affect l-lysine production in the engineered GapN strains, while a drastic decrease in l-lysine production was observed for the control GapA strain. Determination of the intracellular NADPH/NADP(+) ratios revealed that the ratios in the engineered strains were significantly higher than the ratio of the control GapA strain irrespective of the pentose phosphate pathway. These results demonstrate that our strain engineering strategy allows efficient l-lysine production independent of the oxidative pentose phosphate pathway.

Isolation and Characterization of Acetylated Derivative of Recombinant Insulin Lispro Produced in Escherichia coli.

PURPOSE: Insulin lispro is a rapid-acting insulin analogue produced by recombinant DNA technology. As a biosynthetic drug, the protein undergoes strict monitoring aiming for detection and characterization of impurities. The goal of this study was to isolate and identify a derivative of insulin lispro formed during biosynthesis. METHODS: For this purpose, ion exchange chromatography in combination with endoproteinase Glu-C digestion, MALDI-TOF/TOF mass spectrometry and Edman sequencing were employed. RESULTS: Ion exchange chromatography analysis of related proteins in development batches of recombinant insulin lispro revealed the existence of unknown derivative in excess of the assumed limit. Its molecular mass was 42 Da higher than the theoretical mass of Lys(B31) insulin lispro--one of the expected process-related intermediates. Endoproteinase Glu-C cleavage enabled indication of the modified peptide. Tandem mass spectrometry (MS/MS) allowed to explore the location and type of the modification. The 42 amu shift was present in the mass of y-type ions, while b-type ions were in agreement with theoretical values. It suggested that the modification is present on B31 lysine. Further inquiry revealed the presence of two diagnostic ions for lysine acetylation at m/z 143.1 and 126.1. In addition, the peptide was isolated and sequenced by Edman degradation. Standards of phenylthiohydantoin derivatives of N-ε-acetyl-L-lysine and N-ε-trimethyl-L-lysine, not available commercially, were synthesized in the laboratory. The retention time of the modified residue confirmed its identity as N-ε-acetyl-L-lysine. CONCLUSIONS: The derivative of insulin lispro formed during biosynthesis of the drug was identified to be N-ε-acetyl-L-lysine (B31) insulin lispro.

Myxochelins target human 5-lipoxygenase.

Extracts of the predatory myxobacterium Pyxidicoccus fallax HKI 727 showed antiproliferative effects on leukemic K-562 cells. Bioactivity-guided fractionation led to the isolation of the bis-catechol myxochelin A and two new congeners. The biosynthetic origin of myxochelins C and D was confirmed by feeding studies with isotopically labeled precursors. Pharmacological testing revealed human 5-lipoxygenase (5-LO) as a molecular target of the myxochelins. In particular, myxochelin A efficiently inhibited 5-LO activity with an IC50 of 1.9 µM and reduced the proliferation of K-562 cells at similar concentrations.

N-ε-fructosyllysine and N-ε-carboxymethyllysine, but not lysinoalanine, are available for absorption after simulated gastrointestinal digestion.

Food processing leads to a variety of chemical modifications of amino acids in food proteins. Recent studies have shown that some modified amino acids resulting from glycation reactions can pass the intestinal barrier when they are bound in dipeptides. In this study, we investigated as to what extent modified amino acids are released from post-translationally modified casein during simulated gastrointestinal digestion. Casein was enriched with N-ε-fructoselysine, N-ε-carboxymethyllysine, and lysinoalanine, in different degrees of modification. The casein samples were subjected to a two-step proteolysis procedure, simulating gastrointestinal digestion. The digestibility of modified casein as measured by analytical size-exclusion chromatography (SEC) decreased with increasing degree of modification especially after enrichment of fructoselysine and lysinoalanine. Semi-preparative SEC of digested casein samples revealed that fructoselysine and carboxymethyllysine are released bound in peptides smaller than 1,000 Da, which is comparable to native amino acids. The glycation compounds should, therefore, be available for absorption. Lysinoalanine as a crosslinking amino acid, however, is mostly released into longer peptides of at least 30-40 amino acids which should strongly impair its absorption availability.

Increased lysine production by flux coupling of the tricarboxylic acid cycle and the lysine biosynthetic pathway--metabolic engineering of the availability of succinyl-CoA in Corynebacterium glutamicum.

In this study, we demonstrate increased lysine production by flux coupling using the industrial work horse bacterium Corynebacterium glutamicum, which was mediated by the targeted interruption of the tricarboxylic acid (TCA) cycle at the level of succinyl-CoA synthetase. The succinylase branch of the lysine production pathway functions as the bridging reaction to convert succinyl-CoA to succinate in this aerobic bacterium. The mutant C. glutamicum ΔsucCD showed a 60% increase in the yield of lysine when compared to the advanced lysine producer which was used as parent strain. This mutant was highly vital and exhibited only a slightly reduced specific growth rate. Metabolic flux analysis with (13)C isotope studies confirmed that the increase in lysine production was mediated by pathway coupling. The novel strain exhibited an exceptional flux profile, which was closer to the optimum performance predicted by in silico pathway analysis than to the large set of lysine-producing strains analyzed thus far. Fluxomics and transcriptomics were applied as further targets for next-level strain engineering to identify the back-up mechanisms that were activated upon deletion of the enzyme in the mutant strain. It seemed likely that the cells partly recruited the glyoxylate shunt as a by-pass route. Additionally, the α-ketoglutarate decarboxylase pathway emerged as the potential compensation mechanism. This novel strategy appears equally promising for Escherichia coli, which is used in the industrial production of lysine, wherein this bacterium synthesizes lysine exclusively by succinyl-CoA activation of pathway intermediates. The channeling of a high flux pathway into a production pathway by pathway coupling is an interesting metabolic engineering strategy that can be explored to optimize bio-production in the future.

Indirect electrochemiluminescence detection of lysine and histidine separated by capillary electrophoresis based on charge displacement.

Indirect electrochemiluminescence (ECL) detection was applied for the analysis of lysine (Lys) and histidine (His) separated by capillary electrophoresis (CE). With the most effective electrophoretic buffer system, which contained 15 mM phosphate buffer (pH = 5.8) and 0.5 mM Tripropylamine (TPA), fast separation of the two basic amino acids could be performed within 7 min. The linear ranges were 10-35 µM, 35-150 µM for Lys; and 5-35 µM, 35-150 µM for His. The detection limits (S/N = 3) were 0.3 µM for Lys and 1.0 µM for His, respectively. The proposed method was also successfully used for the determination of Lys in the oral pharmaceutical formulations.

The presence of N(ε)-(Carboxymethyl) lysine in the human epidermis.

It is well known that advanced glycation end products (AGEs) are formed in long-lived dermal proteins such as collagen, and that their formation is related to skin aging. To examine the distribution of AGEs in skin tissue, we performed immunofluorescence studies on the human skin using an anti-AGEs antibody. Interestingly, AGEs signals were observed not only in the dermis but also in the epidermis. The objectives of this study were to confirm the presence of N(ε)-(Carboxymethyl) lysine (CML), an AGE structure, in the epidermis and to characterize the CML-modified proteins. The presence of CML in the stratum corneum (SC) was examined using liquid chromatography-electrospray ionization time-of-flight mass spectrometry. Concordance between the retention times of a compound in the SC hydrolysate and authentic CML, as well as with the specific mass transition of CML, was detected. This result showed that CML is present in the epidermis. In order to characterize the CML-modified proteins in the epidermis, protein samples extracted from the SC were analyzed using two-dimensional electrophoresis followed by an amino acid sequence analysis. The clarified peptide sequences covered approximately 27% of the amino acid sequences of cytokeratin 10 (K10). In the immunoblotting experiment following the two-dimensional electrophoresis, where protein samples extracted from whole epidermis were used, the position of the major CML-positive spots corresponded to those of K10. Taken together these results showed that CML is present in the human epidermis, and suggest that K10 is one of the target molecules for CML modification in the epidermis.

A rapid and robust assay for the determination of the amino acid hypusine as a possible biomarker for a high-throughput screening of antimalarials and for the diagnosis and therapy of different diseases.

Eukaryotic initiation factor 5A (eIF5A) has recently been identified as a biomarker of prognostic significance and therapeutic potential for the treatment in hepatocellular carcinoma. This prompted us to establish a rapid and robust assay to determine deoxyhypusine and hypusine formed with the purified enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) from Plasmodium to develop a rapid screening assay for antimalarial drugs. The peptide hydrolysate obtained from hypusinylated eIF5A was analyzed by ultra performance liquid chromatography (UPLC) with retention times for deoxyhypusine of 7.44 min and for hypusine of 7.30 min, respectively. The limit of detection for both compounds was 0.144 ng/µl. Determination of the specific activity of Plasmodium DOHH resulted in a twofold higher specific activity than its human counterpart. Following the iron-complexing strategy of the ferrous iron which is present in the active site of Plasmodium DOHH, a series of iron chelating compounds was tested. 2,2'-Dipyridyl and mimosine abolished DOHH activity completely while 4-oxo-piperidine-carboxylates i.e. the nitrophenylether JK8-2 and EHW 437, the oxime ether of the piperidine aldehyde, showed no inhibition although they were highly active in in vitro cultures of Plasmodium and in vivo in a rodent mouse model. The method allows a high-throughput screening (HPTS) of antimalarial drugs and the evaluation of eIF5A as a biomarker.

Purification and physical properties of group C streptococcal phage-associated lysin.

A purification procedure for the Group C phage-associated lysin is described utilizing tetrathionate to protect the enzyme's -SH group(s) from thiol-inactivating agents. A 652-fold purification has been accomplished yielding a solution in which the enzyme activity corresponds to essentially a single band on polyacrylamide gel which accounts for 70% of the total protein in the preparation. A molecular weight of 101,000 and frictional ratio of 1.526 was determined for the lysin utilizing experimentally determined values for its Stokes radius and sedimentation coefficient.

Partitioning of acidic, basic and neutral amino acids into imidazolium-based ionic liquids.

In this paper, partitioning behaviors of typical neutral (Alanine), acidic (Glutamic acid) and basic (Lysine) amino acids into imidazolium-based ionic liquids [C(4)mim][PF(6)], [C(6)mim][PF(6)], [C(8)mim][PF(6)], [C(6)mim][BF(4)] and [C(8)mim][BF(4)] as extracting solvents were examined. [C(6)mim][BF(4)] showed the best efficiency for partitioning of amino acids. The partition coefficients of amino acids in ionic liquids were found to depend strongly on pH of the aqueous solution, amino acid and ionic liquid chemical structures. Different chemical forms of amino acids in aqueous solutions were pH dependent, so the pH value of the aqueous phase was a determining factor for extraction of amino acids into ionic liquid phase. Both water content of ionic liquids and charge densities of their anionic and cationic parts were important factors for partitioning of cationic and anionic forms of amino acids into ionic liquid phase. Extracted amino acids were back extracted into phosphate buffer solutions adjusted on appropriate pH values. The results showed that ionic liquids could be used as suitable modifiers on the stationary phase of an HPLC column for efficient separation of acidic, basic, and neutral amino acids.

[Studies on the chemical constituents of Teyou 2 Folium Mori].

OBJECTIVE: To study the chemical constituents of Teyou 2 Folium Mori. METHODS: The compounds were extracted and isolated by CO2 supercritical fluid extraction, 30% alcohol extraction, macroporous resin and silica gel column chromatography and their structures were elucidated by means of melting point, TLC and spectral analyses. RESULTS: Six compounds were identified as beta-carotene (I), oleanolic acid (II), benzoic acid (III), chlorogenic acid (IV), lysine (V), gamma-aminobutyric acid (VI). CONCLUSION: Compound II and V are isolated from this plant for the first time.

Tailoring Activated Carbons for Efficient Downstream Processing: Selective Liquid-Phase Adsorption of Lysine.

The essential amino acid lysine is of great importance in the nutrition and pharmaceutical industries and is mainly produced in biorefineries by the fermentation of glucose. In biorefineries, downstream processing is often the most energy-consuming step. Adsorption on hydrophobic adsorbents represents an energy, resource, and cost-saving alternative. The results reported herein provide insights into the selective separation of l-lysine from aqueous solution by liquid-phase adsorption using tailored activated carbons. A variety of commercial activated carbons with different textural properties and surface functionalities is investigated. Comprehensive adsorbent characterization establishes structure-adsorption relationships that define the major roles of the specific surface area and oxygen functionalities. A 13-fold increase of the separation of lysine and glucose is achieved through systematic modification of a selected activated carbon by oxidation, and lysine adsorption is enhanced by 30 %.

Enantioseparation of lysine derivatives on amylose tris (3, 5-dimethylphenylcarbamate) as chiral stationary phase with high separation factor.

Enantioseparation of lysine derivatives by chiral high-performance liquid chromatography (HPLC) was accomplished using two chiral stationary phases (CSPs, Chiralpak IA and Chiralpak IC) based on polysaccharides under normal phase (NP) conditions. All analytes were completely separated. The impacts of polar modifiers, analyte structure and column temperature on chiral separation were discussed. Moreover, the relationship between structure and retention was investigated. The van't Hoff equation was employed to evaluate the thermodynamic parameters of the chiral separation process. The data suggest that the chiral separation process was enthalpy-driven. Surprisingly, two uncommon phenomena were observed: (1) high separation factors on Chiralpak IA and (2) different binding mechanisms with CSP and the two enantiomers.

A microsystem of low-voltage-driven electrophoresis on microchip with array electrode pairs for the separation of amino acids.

In this paper, a new approach for the separation of amino acids on the electrophoresis chip-based low-voltage-driven electrophoresis was reported in detail. This low-voltage-driven electrophoresis process could be realized by powering directly the arrayed electrode pairs with low direct current (DC) voltage to generate a moving electric field along the separation microchannel, which could maintain enough electric field strength for electrophoresis. The proposed microfluidic electrophoresis chip was bonded directly with silicon-on-insulator (SOI) substrate and polydimethylsiloxane (PDMS) cover plate at room temperature. The microfluidic channels and the arrayed electrodes were etched on SOI wafer by silicon microelectromechanical system technology. A specially integrated circuit was proposed to power a 30-60-V DC voltage to particular sets of these electrode pairs in a controlled sequence such that the moving electric field could be formed, and the low-voltage-driven electrophoresis could be realized in the microchannel. In the experiments, with 10(-4) mol/L phenylalanine and lysine as analytes, the separation of amino acids on the low-voltage-driven electrophoresis microchip was conducted by homemade integrated control circuit; a method for separating amino acids was well established. It was also shown that the phenylalanine and lysine mixture was effectively separated in less than 7 min and with a resolution of 2.0. To the best of our knowledge, the low-voltage-driven microchip electrophoresis device could be of potential prospective in the fields of integrated and miniaturized biochemical analysis system.

A suite of asymmetric citrate siderophores isolated from a marine Shewanella species.

Woodybactins A-D are a suite of new fatty acyl siderophores produced by the luminous marine bacterium Shewanella woodyi MS32. While this bacterium has a set of genes homologous to the biosynthetic gene cluster for aerobactin, aerobactin is not produced. The arrangement of these genes within the genome differs in S. woodyi MS32 when compared to E. coli and other species producing siderophores similar to aerobactin, and one synthetase gene which would append a second acyl-hydroxylysine to the terminal carboxylate of citrate is not functional. Within the suite of woodybactins A-D, which differ by the fatty acid appendage, one contains an unusual C9 (9:0) fatty acid and one contains a unique branched C9 iso (9:0 iso) fatty acid, as well as a C8 (8:0) and C10 (10:0) fatty acid.

Determination of N epsilon-(carboxymethyl)lysine in foods and related systems.

The sensitive and specific determination of advanced glycation end products (AGEs) is of considerable interest because these compounds have been associated with pro-oxidative and proinflammatory effects in vivo. AGEs form when carbonyl compounds, such as glucose and its oxidation products, glyoxal and methylglyoxal, react with the epsilon-amino group of lysine and the guanidino group of arginine to give structures including N epsilon-(carboxymethyl)lysine (CML), N epsilon-(carboxyethyl)lysine, and hydroimidazolones. CML is frequently used as a marker for AGEs in general. It exists in both the free or peptide-bound forms. Analysis of CML involves its extraction from the food (including protein hydrolysis to release any peptide-bound adduct) and determination by immunochemical or instrumental means. Various factors must be considered at each step of the analysis. Extraction, hydrolysis, and sample clean-up are all less straight forward for food samples, compared to plasma and tissue. The immunochemical and instrumental methods all have their advantages and disadvantages, and no perfect method exists. Currently, different procedures are being used in different laboratories, and there is an urgent need to compare, improve, and validate methods.

Characterization of MB creatine kinase isoform conversion in vitro and in vivo in dogs.

Time-dependent removal of the COOH-terminal lysine residue from each subunit of tissue MM creatine kinase by plasma carboxypeptidase N produces two additional isoforms that are readily separated, thereby permitting sensitive, early detection of acute myocardial infarction. Only two isoforms of MB creatine kinase have been detected in plasma leading to speculation that the COOH-terminal lysine on the B subunit is resistant to hydrolysis. To define the biochemical changes resulting in MB creatine kinase isoform conversion, we incubated highly purified MB creatine kinase from canine myocardium with plasma carboxypeptidase N. Quantitative anion-exchange chromatography of incubation mixtures and serial plasma samples from dogs subjected to coronary occlusion revealed a second, more acidic form evolved with time that was separated from the tissue isoform. Cyanogen bromide digestion of the two isoforms followed by amino acid sequencing of COOH-terminal peptides showed that MB creatine kinase undergoes removal of the COOH-terminal lysine residue from both M and B subunits. An intermediate form lacking lysine on the M subunit was delineated during incubations by the combined use of anion-exchange chromatography and conventional electrophoretic techniques. Thus, sequential cleavage of lysine from subunits of MB creatine kinase produces an intermediate isoform that has not been detected previously because of difficulties separating it from the tissue and fully converted isoforms.

Effects of ionic speciation of lysine on its adsorption and desorption through a sulfone-type ion-exchange column.

Lysine produced during microbial fermentation is usually recovered by an ion-exchange process, in which lysine is first converted to the cationic form (by lowering the pH to less than 2.0 with sulfuric acid) and then fed to a cation-exchange column containing an exchanger that has a sulfone group with a weak counterion such as NH4+. Ammonia water with a pH above 11 is then supplied to the column to displace the purified lysine from the column and allow its recovery. To enhance the adsorption capacity and for a possible reduction in chemical consumption, monovalent lysine fed at pH 4 was investigated in comparison with conventional divalent lysine fed at pH 1.5. The adsorption capacity increased by more than 70% on a mass basis using pH 4 feeding compared with pH 1.5 feeding. Lysine adsorbed at pH 4 started to elute earlier than that adsorbed at pH 1.5 when ammonia water was used as the eluant solution, and the extent of early elution became more notable at lower concentrations of ammonia. Moreover, the elution of monovalent lysine fed at pH 4 displayed a stiffer front boundary and higher peak concentration. However, when the ammonium concentration was greater than 2.0 N, complete saturation of the bed was delayed during adsorption and the percent recovery yield from elution was lowered, both drawbacks that were considered inevitable features originating from the increased adsorption of monovalent lysine.

Comprehensive analysis of phospholipids and glycolipids in the opportunistic pathogen Enterococcus faecalis.

Enterococcus faecalis is a Gram-positive, opportunistic, pathogenic bacterium that causes a significant number of antibiotic-resistant infections in hospitalized patients. The development of antibiotic resistance in hospital-associated pathogens is a formidable public health threat. In E. faecalis and other Gram-positive pathogens, correlations exist between lipid composition and antibiotic resistance. Resistance to the last-resort antibiotic daptomycin is accompanied by a decrease in phosphatidylglycerol (PG) levels, whereas multiple peptide resistance factor (MprF) converts anionic PG into cationic lysyl-PG via a trans-esterification reaction, providing resistance to cationic antimicrobial peptides. Unlike previous studies that relied on thin layer chromatography and spectrophotometry, we have performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) directly on lipids extracted from E. faecalis, and quantified the phospholipids through multiple reaction monitoring (MRM). In the daptomycin-sensitive E. faecalis strain OG1RF, we have identified 17 PGs, 8 lysyl-PGs (LPGs), 23 cardiolipins (CL), 3 glycerophospho-diglucosyl-diacylglycerols (GPDGDAG), 5 diglucosyl-diacylglycerols (DGDAG), 3 diacylglycerols (DAGs), and 4 triacylglycerols (TAGs). We have quantified PG and shown that PG levels vary during growth of E. faecalis in vitro. We also show that two daptomycin-resistant (DapR) strains of E. faecalis have substantially lower levels of PG and LPG levels. Since LPG levels in these strains are lower, daptomycin resistance is likely due to the reduction in PG. This lipidome map is the first comprehensive analysis of membrane phospholipids and glycolipids in the important human pathogen E. faecalis, for which antimicrobial resistance and altered lipid homeostasis have been intimately linked.