Facile monitoring of avian reovirus σB expression and purification processes by tagged green fluorescent protein

Avian reovirus capsid protein σB was genetically fused with a histidine (His₆) tag and a UV-optimized green fluorescent protein (GFPuv) and expressed in Sf-9 cells. The fluorescence of GFPuv allowed for easy identification of protein localization and revealed that the fusion protein was quite stable in the cell culture. The fluorescence intensity (FI) exhibited a linear relationship (r² = 0.93) with the recombinant protein yield and therefore allowed for on-line tracking of the expression profile, which revealed an extremely high maximum yield of 70 μg per 10⁶ cells. The recombinant protein was purified via immobilized metal affinity chromatography (IMAC) and a high purity (85%) was achieved in one step. During the purification, the fluorescence again enabled qualitative and quantitative monitoring of when and how much the desired product was eluted. The GFP-tagging strategy eliminated the need for cumbersome and time-consuming assays (e.g. Western blot or ELISA) for product analysis, thus GFP is an effective non-invasive on-line marker for the expression and purification of recombinant proteins in the baculovirus expression system.     

A fungal enzyme with the ability of aflatoxin B₁ conversion: purification and ESI-MS/MS identification

Armillariella tabescens, a Chinese edible and medicinal fungus, whose multienzyme exist ability of AFB₁-converting, and ADTZ (aflatoxin-detoxizyme) had previously purified from the A. tabescens multienzyme monitored by AFB₁ conversion_. However, the enzyme now confirmed an oxidase and renamed aflatoxin-oxidase (AFO). In this paper, AFO was purified by an economical and practical three-step procedure monitored by AFB₁ conversion. And ESI-MS/MS analysis was done for identification of AFO. The following database searching (Protein Blast on NCBI) results did not show any homologous oxidase protein, which implied that AFO was mostly a new oxidase differing from other reported aflatoxin-converting enzymes such as fungal laccase and horse radish peroxidase. HPTLC analysis of the purified AFO activity suggested that the enzyme reacted at the bisfuran ring of AFB₁ which was the key toxic structure. Therefore, all these investigations implied a new choice for biodegradation of aflatoxin in foods and feeds with the practical application of AFO.     

Metal binding ability of glutathione transferases conserved between two animal species,the vanadium-rich ascidian Ascidia sydneiensis samea and the schistosome Schistosoma japonicum

Glutathione transferases (GSTs) are multifunctional enzymes found in many organisms. We recently identified vanadium-binding GSTs, designated AsGSTs, from the vanadium-rich ascidian, Ascidia sydneiensis samea. In this study, the metal-selectivity of AsGST-I was investigated. Immobilized metal ion affinity chromatography (IMAC) analysis revealed that AsGST-I binds to V(IV), Fe(III), and Cu(II) with high affinity in the following order Cu(II) > V(IV) > Fe(III), and to Co(II), Ni(II), and Zn(II) with low affinity. The GST activity of AsGST-I was inhibited dose-dependently by not V(IV) but Cu(II). A competition experiment demonstrated that the binding of V(IV) to AsGST-I was not inhibited by Cu(II). These results suggest that AsGST-I has high V(IV)-selectivity, which can confer the specific vanadium accumulation of ascidians. Because there are few reports on the metal-binding ability of GSTs, we performed the same analysis on SjGST (GST from the schistosome, Schistosoma japonicum). SjGST also demonstrated metal-binding ability although the binding pattern differed from that of AsGST-I. The GST activity of SjGST was inhibited by Cu(II) only, as that of AsGST-I. Our results indicate a possibility that metal-binding abilities of GSTs are conserved among organisms, at least animals, which is suggestive of a new role for these enzymes in metal homeostasis or detoxification.     

Nanoengineered analytical immobilized metal affinity chromatography stationary phase by atom transfer radical polymerization: separation of synthetic prion peptides

Atom transfer radical polymerization (ATRP) was employed to create isolated, metal-containing nanoparticles on the surface of nonporous polymeric beads with the goal of developing a new immobilized metal affinity chromatography (IMAC) stationary phase for separating prion peptides and proteins. Transmission electron microscopy was used to visualize nanoparticles on the substrate surface. Individual ferritin molecules were also visualized as ferritin-nanoparticle complexes. The column's resolving power was tested by synthesizing peptide analogs to the copper binding region of prion protein and injecting mixtures of these analogs onto the column. As expected, the column was capable of separating prion-related peptides differing in number of octapeptide repeat units (PHGGGWGQ), (PHGGGWGQ)(2), and (PHGGGWGQ)(4). Unexpectedly, the column could also resolve peptides containing the same number of repeats but differing only in the presence of a hydrophilic tail, Q-->A substitution, or amide nitrogen methylation.     

Characterization of peptides resulting from digestion of human skin elastin with elastase

Several pathological disorders are associated with abnormalities in elastic fibers, which are mainly composed of elastin. Understanding the biochemical basis of such disorders requires information about the primary structure of elastin. Since the acquisition of structural information for elastin is hampered by its extreme insolubility in water or any organic solvent, in this study, human skin elastin was digested with elastase to produce water-soluble peptides. Tandem mass spectrometry (MS/MS) experiments were performed using conventional electrospray ionization (ESI) and nano-ESI techniques coupled with ion trap and quadrupole time-of-flight (qTOF) mass analyzers, respectively. The peptides were identified from the fragment spectra using database searching and/or de novo sequencing. The cleavage sites of the enzyme and, for the first time, the extent and location of proline hydroxylation in human skin elastin were determined. A total of 117 peptides were identified with sequence coverage of 58.8%. It has been observed that 25% of proline residues in the sequenced region are hydroxylated. Elastase cleaves predominantly at the C-terminals of the amino acids Gly, Val, Leu, Ala, and Ile, and to a lesser extent at Phe, Pro, Glu, and Arg. Our results confirm a previous report that human skin elastin lacks amino acid sequences expressed by exon 26A.     

Cloning, expression, purification and characterization of a single chain variable fragment specific to tumor necrosis factor alpha in Escherichia coli

Anti TNF-α molecules have been used as therapeutic agents in a variety of human diseases such as Rheumatoid arthritis, Ankylosing spondylitis, Chron's diseases, Psoriasis, etc., where high levels of TNF-α plays a destructive role. The limitations of the present TNF-α inhibitors in terms of size, tissue penetration and immunogenicity, etc., provoked the search for small anti TNF-α molecules. In the present study, a single chain variable fragment (ScFv) construct was made from a monoclonal antibody of the class IgG raised against TNF-α was used. The anti TNF-α ScFv was well expressed as soluble form in Escherichia coli BL21 (DE3), which was purified to homogeneity by commercial methacrylate monolith-convective interaction media (CIM) supports using two different chemistries, immobilized metal affinity chromatography (IMAC) with copper ions followed by anion exchange chromatography. The anti TNF-α ScFv found to be inhibiting the TNF-α mediated cytotoxicity in MCF-7 cells with an IC₅₀ of 8 μg. Data presented here are promising and encouraging to further optimize anti TNF-α ScFv production in larger scale with higher recovery at a cheaper price for therapeutic purposes.     

Nanoprobe-based immobilized metal affinity chromatography for sensitive and complementary enrichment of multiply phosphorylated peptides

Magnetic nanoparticles (MNP, <100 nm) have rapidly evolved as sensitive affinity probes for phosphopeptide enrichment. By taking advantage of the easy magnetic separation and flexible surface modification of the MNP, we developed a surface‐blocked, nanoprobe‐based immobilized metal ion affinity chromatography (NB‐IMAC) method for the enhanced purification of multiply phosphorylated peptides. The NB‐IMAC method allowed rapid and specific one‐step enrichment by blocking the surface of titanium (IV) ion‐charged nitrilotriacetic acid‐conjugated MNP (Ti⁴⁺‐NTA‐PEG@MNP) with low molecular weight polyethylene glycol. The MNP demonstrated highly sensitive and unbiased extraction of both mono‐ and multiply phosphorylated peptides from diluted β‐casein (2×10^?10 M). Without chemical derivation or fractionation, 1283 phosphopeptides were identified from 400 μg of Raji B cells with 80% purification specificity. We also showed the first systematic comparison on the particle size effect between nano‐sclae IMAC and micro‐scale IMAC. Inductively coupled plasma‐mass spectrometry (ICP‐MS) analysis revealed that MNP had a 4.6‐fold higher capacity for metal ions per unit weight than did the magnetic micro‐sized particle (MMP, 2–10 μm), resulting in the identification of more phosphopeptides as well as a higher percentage of multiply phosphorylated peptides (31%) at the proteome scale. Furthermore, NB‐IMAC complements chromatography‐based IMAC and TiO₂ methods because <13% of mono‐ and 12% of multiply phosphorylated peptide identifications overlapped among the 2700 phosphopeptides identified by the three methods. Notably, the number of multiply phosphorylated peptides was enriched twofold and threefold by NB‐IMAC relative to micro‐scale IMAC and TiO₂, respectively. NB‐IMAC is an innovative material for increasing the identification coverage in phosphoproteomics.     

Inducing and isolation of antibacterial peptides from oriental fruit fly, Bactrocera dorsalis Hendel

One antibacterial activity fraction from an immunized dipteran insect, Bactrocera dorsalis, was isolated and purified by prepurification, ion‐exchange chromatography, gel filtration chromatography and reverse‐phase high performance liquid chromatography (HPLC). The final purified fraction was checked on the Smart system HPLC and was judged as a pure fraction. The results of physical and biological analysis revealed that this fraction is heat stable and showed strong activities against Gram‐positive bacterial growth. It possesses antibicrobial peptide properties and is worth further investigation.     

Increased secretion of latent elastase activity following contact between human skin fibroblasts and elastin derived peptides.

Elastin-derived peptides were previously shown to influence human skin fibroblasts (HSF) chemotaxis and proliferation (Ghuysen et al., 1992). We report here that culturing HSF on κ-elastin (κE) but not onto fibronectin (FN) enhanced the secretion of latent elastinolytic activity. The proteinase involved was identified as the 72 kDa gelatinase A. Moreover, HSF-κE as well as HSF-FN interactions modulated the secretions of Il₁ induced expressions of elastinolytic activities.     

A method for simple identification of signature peptides derived from polyUb-K48 and K63 by MALDI-TOF MS and chemically assisted MS/MS fragmentation

Summary. A simple method is described to identify signature peptides derived from polyubiquitin (polyUb) chains. The method is based on MALDI-TOF MS/MS analysis after chemically assisted fragmentation, and works on peptides isolated from polyacrylamide gels. PolyUb chains branched at K48 and K63 were chosen as models for Ub-protein conjugates. They were resolved by SDS-PAGE, and their tryptic peptides (in-gel-trypsinolysis) derivatized with 3-sulfopropinic acid NHSester to obtain chemically assisted fragmentation during the MS/MS analysis. PolyUb-K63 produced a single peptide identified as ⁵⁵TLSDYNIQK⁶³ (GG)ESTLHLVLR⁷². PolyUb-K48 produced two branched signature peptides identified as ⁴³LIFAGK⁴⁸(GG)QLEDGR⁵⁴ and ⁴³LIFAGK⁴⁸(LRGG)QLEDGR⁵⁴. The recovery of signature peptide with LRGG as branched chain underscores the need to take limited proteolysis into account in the search for detection of ubiquitinated peptides in proteomics studies. In conclusion, a simple method has been described allowing the identification of signature peptides, which are diagnostic markers of the majority of polyUb-conjugated proteins. In principle, the method should be applicable also for other more rare signature peptides.     

Capture of an activated receptor complex from the surface of live cells by affinity receptor chromatography

Cell surface receptors and their associated signaling pathways on the plasma membrane are key targets in understanding cellular responses. However, the isolation and identification of receptor complexes has been elusive. The Fc receptor was captured from the surface of live cells using microbeads coated with the receptor’s cognate ligand, gamma globulin (IgG), and analyzed by liquid chromatography and tandem mass spectrometry (LC–MS/MS) alongside several controls. Live-cell affinity receptor chromatography (LARC) resulted in a partially nonredundant list of 288 proteins that were specific to the Fc receptor complex. The proteins identified were in close agreement with previously determined factors in the Fc receptor complex as demonstrated by genetic and biochemical methods and revealed novel complex members. Confocal microscopy was used to confirm recruitment of SRC, SYK, PLC, PKC, PI3K, SHIP, TEC, CDC42, RAP, PAK, GAP, GEF, GRP, and CRK to the receptor complex upon activation by the same ligand microbeads. The expression of mutants and silencing RNA against specific isoforms were used to demonstrate a functional role for novel members of the Fc receptor complex, including RHOG (RAS homologue member G), p115 RhoGEF (protein of 115-kDa RAS homologue guanine exchange factor), and CRKL (CRK-like). The recruitment of AKT pleckstrin homology (PH) domain green fluorescent protein (GFP) was used to quantify the production of phosphorylated inositol at the activated receptor complex. We conclude that it is feasible to capture an activated receptor complex from the surface of live cells using ligand-coated microbeads for identification of members of a receptor complex or pathway by LC–MS/MS.     

Chemical Modification of Hamster Arylamine N-Acetyltransferase 2 with Isozyme-Selective and Nonselective N-Arylbromoacetamido Reagents

Arylamine N-acetyltransferases (NATs) catalyze a variety of biotransformation reactions, including N-acetylation of arylamines and O-acetylation of arylhydroxylamines. Chemical modification of hamster recombinant NAT2 with 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide revealed that Br-AAF is an affinity label for the enzyme whereas bromoacetanilide inactivates NAT2 through a bimolecular alkylation process. Electrospray ionization quadrupole time-of-flight mass spectrometry analysis of Br-AAF-treated NAT2 showed that a single molecule of 2-acetylaminofluorene had been adducted. Peptide sequencing with tandem mass spectrometry identified the catalytically essential Cys68 as the alkylated amino acid. Br-AAF exhibits similar affinity for hamster NAT1 and NAT2, but is a more effective inactivator of NAT1 because, subsequent to the formation of a reversible enzyme-Br-AAF complex, the rate of alkylation of NAT1 is greater than the rate of alkylation of NAT2. Bromoacetanilide alkylates Cys68 and, to a lesser extent, Cys237 of NAT2; it does not exhibit significant selectivity for either NAT1 or NAT2.     

Speciation of coagulase negative staphylococci, isolated from indoor air, using SDS PAGE gel bands of expressed proteins followed by MALDI TOF MS and MALDI TOF-TOF MS-MS analysis of tryptic peptides

A group of coagulase negative staphylococcal strains isolated from indoor air of occupied school rooms were the subject of this study. Conventional MALDI TOF MS profiling of cellular extracts and physiological tests (including API STAPH) provided incomplete identification of the set of strains. After separation of a 100 kDa band using 1D gel electrophoresis, profiling of peptides (released with tryptic digestion) using MALDI TOF MS allowed improved bacterial speciation in addition to determination of the identity of the protein of origin (aconitate hydratase). This was performed by Mascot search, empirical observation and computer-generated cross-correlation analysis of environmental isolates versus reference strains. The species studied included some with sequenced genomes and others with un-sequenced genomes. Peptide sequences were confirmed to originate from aconitate hydratase using MALDI TOF-TOF MS-MS analysis of a diverse set of m/z values representing variable and conserved sequences. The methodological approach described here might have widespread application in speciation of environmental isolates of diverse origin and in identification of their expressed proteins.     

Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS

Blood-borne biomarkers are urgently required for the early detection, accurate diagnosis and prognosis of disease. Additionally, improved methods of profiling serum and plasma proteins for biomarker discovery efforts are needed. Herein, we report a quantitative method based on amino-group labelling of serum proteins (rather than peptides) with isobaric tandem mass tags (TMT) and incorporating immune-based depletion, gel-based and strong anion exchange separation of proteins prior to differential endoproteinase treatment and liquid chromatography tandem mass spectrometry. We report a generally higher level of quantitative coverage of the serum proteome compared to other peptide-based isobaric tagging approaches and show the potential of the method by applying it to a set of unique samples that pre-date the diagnosis of pancreatic cancer.     

Characterization of antimicrobial peptides isolated from the skin of the Chinese frog, Rana dybowskii

The skins of amphibians secrete small antimicrobial peptides that fight infection and are being explored as potential alternatives to conventional antibiotics. In this study we combined mass spectrometry with cDNA sequencing to examine antimicrobial peptides in skin secretions from the Chinese frog Rana dybowskii. Thirteen peptides having precursor sequences that resemble known antimicrobial peptides from this genus were identified, ten of which were members of previously described peptide families based on their primary structures; i.e., brevinin-1, Japonicin-1, brevinin-2 and temporin. The other three peptides from R. dybowskii, which were named dybowskin-1CDYa, dybowskin-2 CDYa and dybowskin-2CDYb, had different amino acid compositions and little sequence similarity to known antimicrobial peptides. The carboxyl terminus of dybowskin-1CDY lacked amidation and is therefore clearly distinct from temporin peptides, whereas dybowskin-2CDYa and dybowskin-2CDYb consisted of 18 amino acids and were rich in Arg residues. Chemically synthesized peptides corresponding to mature dybowskin-1CDYa and dybowskin-2CDYa had strong antimicrobial activity and caused little hemolysis of human erythrocytes, suggesting they may serve as interesting templates for the development of novel antibiotics.     

Possible Evidence of Contamination by Catechins in Deconjugation Enzymes from Helix pomatia and Abalone entrails

β-Glucuronidase and sulfatase are the major deconjugating enzymes used in the cleavage of the glucuronate and sulfate moieties, respectively, from certain conjugated food factors including polyphenols. In the present study, we found that compounds having the same molecular weights as catechins were present in Helix pomatia- and/or Abalone entrails-derived β-glucuronidase and sulfatase by liquid chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring methods. On the other hand, the same molecular weights as catechins were undetectable in Escherichia coli-derived β-glucuronidase and Aerobacter aerogenes-derived sulfatase. By high performance liquid chromatography, enzyme-derived catechins were not detected because of approximately 1,000-fold lower sensitivity as compared to LC-MS/MS. These results suggest that the catechins in these enzymes might be attributed to the diets of the organisms as the enzyme sources.     

Phosphorylation site mapping of soluble proteins: bioinformatical filtering reveals potential plastidic phosphoproteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Protein phosphorylation is a major mode of regulation of metabolism, gene expression, and cell architecture. A combination of phosphopeptide enrichment strategies based on TiO₂ and IMAC in addition to our MudPIT strategy revealed the detection of 181 phosphorylation sites which are located on 125 potentially plastidic proteins predicted by GoMiner, TargetP/Predotar in Arabidopsis thaliana. In our study phosphorylation on serine is favored over threonine and this in turn over phosphorylation on tyrosine residues, showing a percentage of 67.4% to 24.3% to 8.3% for pS:pT:pY. Four phosphorylated residues (S208, Y239, T246 and T330), identified by our approach have been fitted to the structure of the activated form of spinach RuBisCO, which are located in close proximity to the substrate binding site for ribulosebisphosphate. Potentially, these phosphorylation sites exert a direct influence on the catalytic activity of the enzyme. Such examples show nicely the value of the presented mass spectrometric dataset for further biochemical applications, since alternative mutation analysis often turns out to be unsuccessful, caused by mutations in essential proteins which result in lethal phenotypes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.