Peptide Mass Fingerprinting Analysis Research Articles

Validation of biphenyl degradation pathway by polymerase chain reaction, peptide mass fingerprinting and enzyme analysis

Our previous studies showed, bacterium Aquamicrobium sp. SK-2 could degrade biphenyl and polychlorinated biphenyls (PCBs). In the present study, proteins involved in the biphenyl degradation was evaluated using various molecular biology methods. The gene bphC present in the strain SK-2 was identified using the polymerase chain reaction method. Further the key enzyme in biphenyl degradation, 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) was purified through anion exchange and gel filtration chromatography, subsequently the enzyme activity was measured. The N-terminal amino acid sequence of the purified enzyme showed 92% homology with BphC enzyme of Gram-negative bacteria (Pseudomonas sp. KKS102, Comamonas testosterone, Burkholderiaceae bacterium, Delftia acidovorans, and Achromobacter denitrificans). Fractions collected during protein purification were applied on SDS-PAGE gel. Significant bands were selected in SDS-PAGE gel, and the gel pieces were cut out to analyze the proteins using peptide mass fingerprinting (PMF) method. PMF method provided useful information about the proteins involved in biphenyl degradation. Apart from BphC, two other enzymes, benzoate dioxygenase and catechol 2,3-dioxygenase which were involved in biphenyl degradation process were identified. The results indicate that catechol can be degraded to 2-hydroxymuconic-semialdehyde and this result is in accordance with the results from our previous study. Based on all these results we can conclude that the strain SK-2 is a potential candidate for the bioremediation of biphenyl contaminated places.

Acinetobacter baumannii as a community foodborne pathogen: Peptide mass fingerprinting analysis, genotypic of biofilm formation and phenotypic pattern of antimicrobial resistance.

Acinetobacter baumannii (A. baumannii) is one of the most common Gram-negative pathogens that represent a major threat to human life. Because the prevalence of Multidrug-resistant biofilm-forming A. baumannii is increasing all over the world, this may lead to outbreaks of hospital infections. Nonetheless, the role of raw meat as a reservoir for A. baumannii remains unclear. Here our research was aimed to exhibit the frequency, precise identification, and genotyping of biofilm-related genes as well as antimicrobial resistance of A. baumannii isolates of raw meat specimens. Fifty-five A. baumannii strains were recovered from 220 specimens of different animal meat and then identified by Peptide Mass Fingerprinting Technique (PMFT). All identified isolates were genotyped by the qPCR method for the existence of biofilm-related genes (ompA, bap, blaPER-1, csuE, csgA, and fimH). In addition, the antimicrobial resistance against A. baumannii was detected by the Kirby-Bauer method. Based on our findings, the frequency rate of 55 A. baumannii isolates was 46.55%, 32.50%, 15.00%, and 9.68% of sheep, chicken, cow, and camel raw meat samples, respectively. The PMFT was able to identify all strains by 100%. the percentages of csuE, ompA, blaPER-1, bap, and csgA genes in biofilm and non-biofilm producer A. baumannii were 72.73%, 60%, 58.2%, 52.74%, and 25.45%, respectively. In contrast, the fimH was not detected in all non-biofilm and biofilm producer strains. The ompA, bap, blaPER-1, csgA were detected only in biofilm-producing A. baumannii isolates. The maximum degree of resistance was observed against amoxicillin/clavulanic acid (89.10%), gentamicin (74.55%), tetracycline (72.73%), ampicillin (65.45%), and tobramycin (52.73%). In conclusion, our investigation demonstrated the high incidence of multi-drug resistant A. baumannii in raw meat samples, with a high existence of biofilm-related virulence genes of ompA, bap, blaPER-1, csgA. Therefore, it has become necessary to take the control measures to limit the development of A. baumannii.

Room‐Temperature Synthesis of Trypsin‐Inorganic Hybrid Nanocomposites for Fast and Efficient Protein Digestion

AbstractFast and high‐efficiency protein digestion is essential for high‐throughput proteome analysis. Herein, a new kind of trypsin‐inorganic hybrid nanocomposites were room‐temperature synthesized based on self‐assembly stragety, where trypsin and nickel phosphate (Ni3(PO4)2) was used as organic and inorganic ligands, respectively. The general applicability of the synthetic method was also confirmed by using a series of other metal phosphates including Mg2+, Ba2+, Zn2+, and Co2+. The as‐prepared trypsin‐inorganic hybrid nanocomposites with flower‐like morphology showed higher enzymatic acticity (206 % enhancement) and faster catalytic kinetics than the free trypsin. Taking the merits togethers as mentioned above, the hybrid nanocomposites could be employed as an immmobilized enzyme reactor (IMER) for fast and high‐efficiency protein digestion. The peptide mass fingerprint analysis of the model proteins demonstrated that the coverage rate of the sequences treated by IMER could bear comparsion with that of the free one, but the digestion time was shorten from 12 h to 1 min. In addition, the IMER showed a good applicability for complex human serum sample, showing a promising potential in proteomics research.

An improved protocol for large scale production of high purity ‘Fc’ fragment of human immunoglobulin G (IgG-Fc)

We describe a simplified approach for purification and characterization of human ‘IgG-Fc’ fragment used widely as immunochemical tool and for therapeutic purposes. The ‘Fc’ fragment was purified from human IgG in a 3-stage column chromatography. The purified ‘Fc’ fragment appeared as a dimer glycoprotein with an apparent molecular mass of 52,981 Dalton (Ultraflex MALDI TOF/TOF). The Size-exclusion HPLC profile of the purified ‘Fc’ fragment of human IgG matched that of a commercially procured reference ‘Fc’ fragment material. The purity of the ‘Fc’ fragments was >99% by SDS-PAGE and size-exclusion HPLC. The results of Western blotting, immunoelectrophoresis, and mass spectrometry analysis indicate a high purity of the ‘Fc’ fragment. Peptide mass fingerprint analysis of the purified ‘Fc’ protein yielded peptides that partially match the known database sequences of FCG3B_HUMAN (Uniprot ID: O75015). This method of purification of the ‘Fc’ fragment is suitable for achieving high purity level of ‘Fc’ fragment protein. With this purification approach, the cost of the purified ‘Fc’ fragment of human IgG is significantly reduced as compared with the current market price of IgG-Fc fragment protein in international market. The purified ‘IgG-Fc’ fragment protein was found to be negative for major viral markers.

Proteomic profiles of unilateral cryptorchidism in pigs at different ages using MALDI-TOF mass spectrometry and in-gel digestion coupled with mass spectrometry (GeLC-MS/MS) approaches

BackgroundCryptorchidism is a condition that occurs when one or both testes fail to descend into the scrotum. It is a common congenital disorder, causing economic loss in pig production. However, there have been only limited studies of differential protein expression profiles in undescended testes (UDTs) in the abdomen and descended testes (DTs) in cryptorchid pigs, especially at the peptidome and proteome levels. The present study aimed to analyze the peptidome of UDTs and DTs in unilateral cryptorchid pigs aged 1–2, 6, 15 and 20 weeks and in normal testes of healthy pigs aged 1–2 and 12 weeks, using peptide mass fingerprinting and three-dimensional principal component analysis (3D-PCA) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and to identify potential protein candidates, using in-gel digestion coupled with mass spectrometry (GeLC-MS/MS). Western blot analysis was used to verify protein expression. Protein sequence was affirmed by liquid chromatography–tandem mass spectrometry.ResultsA PCA plot showed a discrete cluster for each sample group. Peptide mass fingerprints (PMFs) demonstrated unique peptide fragments in UDTs at different ages. A number of markedly expressed proteins from GeLC-MS/MS were identified, including the multifunctional tumor necrosis factor receptor superfamily member 18 (TNFRSF18), in DTs at 1–2 and 6 weeks and in UDTs at 15 and 20 weeks of age. Using western blot analysis, high expression of TNFRSF18 was observed in the UDTs at 15 weeks. Using the STITCH database, this protein was found to be related to apoptosis, corresponding to the previous report in the UDTs at the same age.ConclusionsThe present study revealed the specific PMFs and clusters for porcine cryptorchidism, and a novel protein, TNFRSF18, associated with the disease mechanism. These results could provide further insights into the pathogenesis of the disease.

Structure of intact chitinase with hevein domain from the plant Simarouba glauca, known for its traditional anti-inflammatory efficacy

Chitinase from the leaves of Simarouba glauca, a plant used in traditional anti-inflammatory therapy is purified and characterized. Peptide mass finger print analysis revealed the protein as an endo-chitinase which was further confirmed using chitin-agar assay. The enzyme exhibited significant anti-fungal efficacy against phyto-pathogens such as Macrophomina phaseolina, Fusarium oxysporum and Sclerotium rolfsii. Chitinolysis was also examined against insoluble chitin using SEM. Using X-ray diffraction data up to 1.66 Å, the structure was determined by Molecular Replacement using crystal structure of GH19 Chitinase-like protein from Hevea brasiliensis. During structure refinement, an extra domain could be traced and identified as hevein domain. To our knowledge, this is the first report of any chitinase with intact hevein domain. The GH19 chitinase and hevein domains though connected by a lengthy loop, are restricted to be close by disulfide bridges. These bridges connecting each domain with the loop may be important for proper chitin feeding into the active site. By considering reports on hevein and chitinase domains as well as the traditional use of the plant, this report of an intact hevein-chitinase protein and their relative orientation may add further insights for the usefulness of this protein.

Role of arginine kinase in Paramecium tetraurelia (Ciliophora, Peniculida): Subcellular localization of AK3 and phosphoarginine shuttle system in cilia

The ciliate Paramecium tetraurelia has four arginine kinase genes (AK1, AK2, AK3, and AK4). Of these genes, only AK3 has a signal sequence for farnesylation, a post-translational modification that enables anchoring of the modified enzyme to the ciliary membrane. To confirm this modification, AK3 was synthesized using a cell-free protein synthesis system and the peptide masses were analyzed using peptide mass fingerprinting (PMF). The PMF analysis indicated that the C-terminal peptide of AK3 is farnesylated. Thus, AK3 can be farnesylated under physiologically appropriate conditions. To determine the subcellular localization of P. tetraurelia AK3, Western blot analysis was performed using an AK3 polyclonal antibody for the proteins extracted from intact cells and ciliary fractions. When extraction was performed using Triton X-100, AK3 was detected the ciliary fraction. This result suggested that the ciliary fraction contains AK3. In addition, we investigated the role of P. tetraurelia AKs in ciliary movement using the feeding RNA interference method. The swimming velocity of AK1- and AK3-silenced cells was significantly reduced to half the value of that control cells. In summary, P. tetraurelia AK3 is likely to be located in the ciliary membrane and influences swimming velocity, presumably through the phosphoarginine shuttle system present in cilia.

Minimally Invasive Sampling of Surface Coatings for Protein Identification by Peptide Mass Fingerprinting: A Case Study with Photographs

ABSTRACT This study investigates the efficacy of a sampling technique to acquire sufficient sample for peptide mass fingerprinting analysis (PMF) with minimal alteration to photograph surfaces. The technique, which is potentially useful for sampling surface coatings in a wide variety of situations, uses very fine polishing film (1–30 μm particles, 14,000–600 grit) to abrade and remove small amounts of surface material consistent with PMF sample requirements. Several variations of sampling devices were evaluated using coated salt print mock-ups and study collection photographs. This paper discusses those evaluations, proposes an optimized system for sampling thin, proteinaceous coatings for PMF analysis, and cites criteria for deciding whether using the sampling technique is warranted and/or advisable in certain cases.

Purification and Characterization of α-amylase from a Novel Thermoalkalophilic Strain of Bacillus sonorensis GV2 Isolated from Mushroom Compost

A novel thermoalkalophilic α-amylase producing bacterial strain Bacillus sonorensis GV2 |KJ775811.1| was isolated from mushroom compost. The purification of α-amylase was performed through different chromatography techniques. After purification with SDS-PAGE and Sephadex G-75 gel filtration, the molecular weight of monomeric α-amylase was found to be 45 kDa. The enzyme showed an optimal activity over a wide range of temperature and pH of 35-60oC and 7-11, respectively. The effect of divalent ions i.e. Mg2+ and Ca2+ showed a positive increase in enzyme activity. This enzyme is unique in a sense that it also exhibited a considerable raw corn starch hydrolyzing activity at 55oC. The end products when subjected to TLC which were identified as main maltooligosaccharides, proving the endo action of an enzyme. The Vmax and Km values of Bacillus sonorensis GV2 α-amylase were found to be 1347 μmol/mg/min and 3.46 mMol/ml. The MALDI peptide mass fingerprint analysis of the reduced and carboxymethylated amylase digested with chymotrypsin indicated that this partial amino acid sequence was homologous by a score of 6 with UDP transferalyase. All these findings suggests about the potential role of this α-amylase for raw starch degrading applications in the relevant industry.

Novel Victorivirus from a Pakistani Isolate of Alternaria alternata Lacking a Typical Translational Stop/Restart Sequence Signature.

The family Totiviridae currently contains five genera Totivirus, Victorivirus, Leishmavirus, Trichomonasvirus, and Giardiavirus. Members in this family generally have a set of two-open reading frame (ORF) elements in their genome with the 5′-proximal ORF (ORF1) encoding a capsid protein (CP) and the 3′-proximal one (ORF2) for RNA-dependent RNA polymerase (RdRp). How the downstream open reading frames (ORFs) are expressed is genus-specific. All victoriviruses characterized thus far appear to use the stop/restart translation mechanism, allowing for the expression of two separate protein products from bicitronic genome-sized viral mRNA, while the totiviruses use a −1 ribosomal frame-shifting that leads to a fusion product of CP and RdRp. We report the biological and molecular characterization of a novel victorivirus termed Alternaria alternata victorivirus 1 (AalVV1) isolated from Alternaria alternata in Pakistan. The phylogenetic and molecular analyses showed AalVV1 to be distinct from previously reported victoriviruses. AalVV1 appears to have a sequence signature required for the −1 frame-shifting at the ORF1/2 junction region, rather than a stop/restart key mediator. By contrast, SDS–polyacrylamide gel electrophoresis and peptide mass fingerprinting analyses of purified virion preparations suggested the expression of two protein products, not a CP-RdRp fusion product. How these proteins are expressed is discussed in this study. Possible effects of infection by this virus were tested in two fungal species: A. alternata and RNA silencing proficient and deficient strains of Cryphonectria parasitica, a model filamentous fungus. AalVV1 showed symptomless infection in all of these fungal strains, even in the RNA silencing deficient C. parasitica strain.

Identification of a binding protein for sesamin and characterization of its roles in plant growth

Sesamin is a furofuran-type lignan that is found abundantly in seeds of Sesamum indicum (sesame) and has been widely accepted as a dietary supplement with positive effects on human health. The biological activity of sesamin in human cells and organs has been analysed extensively, although comparatively few studies show biological functions for sesamin in planta. Herein we screened sesamin-binding proteins (SBP) from sesame seedling extracts using sesamin-immobilized nano-beads. In subsequent peptide mass fingerprinting analyses, we identified a SBP, Steroleosin B, which is one of the membrane proteins found in oil bodies. In addition, pull-down assays and saturation transfer difference-nuclear magnetic resonance (STD-NMR) experiments demonstrated that sesamin binds directly to recombinant Steroleosin B in vitro. Finally, ectopic accumulations of sesamin and Steroleosin B in transgenic Arabidopsis thaliana plants induced severe growth defects including suppression of leaf expansion and root elongation. Collectively, these results indicate that sesamin influences tissue development in the presence of Steroleosin B.

A cost-effective method for purification and characterization of human urinary albumin

We describe a simplified approach for the purification and characterization of urinary albumin, a key biomarker currently used for understanding the onset and prognosis of microalbuminuria. Urinary albumin was purified from human urine collected from diabetic kidney disease patients by using 2-stage tangential flow filtration process and set of column chromatography steps. The relative molecular mass of urinary albumin is 66,871 Da (SYNAPT G2 High Definition Mass Spectrometry System). Isolated urinary albumin was analyzed by SDS-PAGE, Western blotting, immunoelectrophoresis, Ouchterlony double-immunodiffusion, single radial immunodiffusion, size-exclusion HPLC and peptide mass fingerprint analysis. The size-exclusion HPLC elution profile of the purified urinary albumin was similar to that of a reference form of native albumin. Peptide mass fingerprint analysis of the purified urinary albumin yielded peptides that partially matched with known sequence of ALBU_HUMAN (P02768). This is the first report of purification and validation of immunochemically reactive form of urinary albumin from a large volume of urine of diabetic kidney disease patients. In this purification approach, the cost of the purified albumin is significantly lower.

Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea

Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216 Da) and RsKI (19,412 Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki = 128.5 ± 4.5 nM) and chymotrypsin (Ki = 807.8 ± 23.7 nM) while RsKI (Ki = 172.0 ± 9.2 nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100 °C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100 °C and retained >60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > β-sheets/β-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75 °C. Further, the significant inhibitory activity of RsBBI (IC50 = 24 ng) and RsKI (IC50 = 59 ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively.

Identification of human plasma C1 inhibitor as a target protein for staphylococcal superantigen-like protein 5 (SSL5)

The family of staphylococcal superantigen-like proteins (SSLs) have a structure similar to bacterial superantigens but exhibit no superantigenic activity. These exoproteins have recently been shown to disturb the host immune defense system. One family member, SSL5, was reported to bind to human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase-9 (MMP-9) and to interfere with leukocyte trafficking. In the present study, we explored human plasma proteins bound by glutathione S-transferase (GST)-tagged recombinant SSL5 (GST-SSL5) and identified plasma protease C1 inhibitor (C1Inh) as a major SSL5-binding protein based on the results of peptide mass fingerprinting analysis with MALDI-TOFMS. GST-SSL5 was found to attenuate the inhibitory activity of recombinant histidine-tagged C1Inh (C1Inh-His) toward complement C1s. We also observed that the treatment of C1Inh-His with neuraminidase markedly decreased its binding to GST-SSL5. Moreover, C1Inh-His produced by Lec2 mutant cells (deficient in sialic acid biosynthesis) showed much lower binding affinity for SSL5 than that produced by the wild-type CHO-K1 cells, as assessed by pull-down assay. These results suggest that SSL5 binds to C1Inh in a sialic acid-dependent fashion and modulates the host immune defense through perturbation of the complement system in association with S. aureus infection.

Peptidomic characterization and bioactivity of Protoiurus kraepelini (Scorpiones: Iuridae) venom.

Protoiurus kraepelini is a scorpion species found in parts of Turkey and Greece. In this study, the peptide profile of its venom was determined for the first time. The electrophoretic profile of the crude venom showed a protein distribution from 2 to 130 kDa. MALDI-TOF MS analysis of the venom peptide fraction yielded 27 peptides between 1059 and 4623 Da in mass. Several ion channelblocking and antimicrobial peptides were identified by peptide mass fingerprinting analysis. Cytotoxic and antimicrobial effects of the venom were also demonstrated on Jurkat cells and Escherichia coli, respectively. As the first peptidomic characterization study on P. kraepelini venom, this report lays the foundation for detailed future studies that may lead to the discovery of novel bioactive peptides.

Structural and functional comparability study of anti-CD20 monoclonal antibody with reference product.

BackgroundCell surface protein, CD20, is extensively expressed on the surface of B cells. Antibodies targeting CD20 protein are being used to treat B-cell malignancies and B-cell mediated autoimmune diseases. Considering the cost of therapy with innovator monoclonal antibodies for these diseases, development of biosimilar products for the treatment of such diseases provides affordable solution to rising healthcare costs.Materials and MethodsReference products of rituximab (six batches) were procured and stored as per manufacturer's instructions. Cell lines used in bioassay were procured from American Type Culture Collection and all other reagents used for analysis were of analytical grade. Primary structure was studied by intact mass analysis, peptide fingerprinting, peptide mass fingerprinting and sequence coverage analysis. Higher order structure was studied by circular dichroism, ultraviolet-visible spectroscopy, fluorescence spectroscopy, and disulfide bridge analysis. Different isoforms of reference product and SB-02 were identified using capillary isoelectric focusing and capillary zone electrophoresis. Glycosylation was studied by N-glycan mapping using LC-ESI-MS, point of glycosylation, released glycan analysis using ultra performance liquid chromatography (UPLC). Product related impurities such as oligomer content analysis and oxidized impurities were studied using size exclusion chromatography and reverse phase high performance liquid chromatography, respectively.Results and ConclusionHere, we report physicochemical and biological characterizations of Sun Pharma’s proposed biosimilar (SB-02) to rituximab, a monoclonal anti-CD20 antibody approved for the treatment of non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. SB-02 and rituximab exhibited indistinguishable primary as well as higher-order structure upon analyzing with the array of analytical and extended characterization methods according to statistical methods. The molecule also displayed comparability to reference product in post-translational modifications and charge heterogeneity. In functional bioassays, SB-02 demonstrated comparable potency with respect to reference product. Our results indicate highly similar quality profile between SB-02 and rituximab.

Tissue-specific and intracellular localization of indican synthase from Polygonum tinctorium

The plant Polygonum tinctorium produces the secondary metabolite indican (indoxyl-β-D-glucoside), a precursor of the blue dye indigo. P. tinctorium synthesizes indican through the actions of the UDP-glucosyltransferase (UGT), indican synthase. Herein, we partially purified an indican synthase from the leaves and subsequently performed peptide mass fingerprinting analysis. Consequently, we identified a fragment that was homologous to a UDP-glucosyltransferase 72B (UGT72B) family member. We named it PtIgs (P. tinctoriumindoxyl-β-D-glucoside synthase) and obtained the full-length cDNA using rapid amplification of the cDNA ends. The primary structure of PtIGS, which PtIgs encoded, showed high identity with indican synthases (ItUGT1 and ItUGT2) from Indigofera tinctoria (Inoue et al., 2017). Moreover, in expression analyses of P. tinctorium, PtIGS mRNA was virtually found only in the leaves, was most highly expressed in the 1st leaves, and decreased with leaf age. Because PtIGS expression tended to reflect indican contents and synthesis activities, we concluded that PtIGS functions as an indican synthase in plant cells.To examine intracellular localization of PtIGS, crude leaf extracts were separated into cytosol and microsome fractions, and found PtIGS in the cytosol and in microsome fractions. Furthermore, microsomal PtIGS was soluble in the presence of detergents and urea and was strongly associated with membranes. Finally, we confirmed endoplasmic reticulum (ER) membrane localization of PtIGS using ultracentrifugation with a sucrose density gradient. These data suggest that PtIGS interacts with some kind of proteins on ER membranes to certainly carry out a delivery of substrate.

Chemometrics-Assisted Shotgun Proteomics for Establishment of Potential Peptide Markers of Non-Halal Pork (Sus scrofa) among Halal Beef and Chicken

Authentication of meat is a global concern owing to meat adulteration with pork; however, no study, to date, has established a method to identify species-specific peptide markers for ascertaining adulterated meat. The present study aimed to describe chemometrics-assisted shotgun proteomics, which has recently been reported to establish potential peptide markers for non-Halal pork among Halal beef and chicken meat. To identify the potential peptide markers through peptide mass fingerprinting (PMF) analysis, chemometric analysis comprised principal component analysis and orthogonal partial least square-discriminant analysis, both of which proved statistically useful. Subsequently, through targeted tandem liquid chromatography-mass spectrometry (LC-MS) analysis, the primary structure of the identified peptide markers was revealed using the de novo identification approach. A decoy, randomised and concatenated database search program comprising MS-Fit and MS-Tag was used for PMF and targeted tandem LC-MS analysis, respectively. Results obtained using both PMF and targeted tandem LC-MS analysis complemented one another, indicating that these methods can yield consistent results to identify peptide markers together with chemometric analysis.

PO-062 BAX and BAK interaction with the mitochondrial permeability transition pore (MPTP) is required for taxol-mediated apoptosis

IntroductionMicrotubule Interfering Agents (MIA’s), such as Taxol (Paclitaxel) are used in the treatment of cancers such as breast, ovarian and non–small cell lung cancer. MIA’s arrest cells in mitosis by activating the mitotic checkpoint. Prolonged mitotic arrest results in apoptotic cell death although the precise mechanism of Taxol-induced cell death is unclear. Previous studies have shown that Taxol activates the pro-apoptotic effector proteins, Bak and Bax, which accumulate at the mitochondrial outer membrane. At the mitochondrion Bax and Bak are thought to oligomerize and/or interact with the Mitochondrial Permeability Transition Pore (MPTP) to increase the permeability of the mitochondrial outer membrane which then leads to cytochrome c release and apoptosis. In this study we have examined the requirement for Bax and Bak in Taxol-induced apoptosis and their possible interaction with components of the MPTP.Material and methodsHuman cervical carcinoma (HeLa) cells were treated with either Bax, Bak or both Bax and Bak siRNA, synchronised and treated with Taxol (60 nM) for varying times (0–24 hour). Apoptosis was assessed using either cytokeratin 18 cleavage (M30 antibody), poly (ADP-ribose) polymerase (PARP) cleavage or activation of pro-caspases (3 and 9). Bak and Bax were immunoprecipitated using anti-active Bak (N-20) and anti-active Bax (6A7) antibodies in CHAPS lysis buffer. To identify interacting proteins, Bak and Bax IPs were subjected to peptide mass fingerprint analysis by mass spectrometry. Confocal microscopy was used to examine the intracellular localization of Bax and Bak following Taxol treatment.Results and discussionsThe results of our siRNA studies indicate that although Bax and Bak can form homo-oligomers in the absence of each other, both proteins are required for Taxol-induced apoptosis. Our immunofluorescence study shows that Bak and Bax co-localise at mitochondria in the Taxol-arrested cells. Our proteomic and co-IP analyses indicate that Bak and Bax form a complex specifically in the Taxol-arrested cells that also includes components of the MPTP such as the voltage dependent anion channel (VDAC), the adenine nucleotide translocator (ANT2) and Bcl-2.ConclusionWe conclude that the oligomerisation of Bax and Bak is insufficient for Taxol-mediated apoptosis. However, the interaction of activated Bax and Bak with the MPTP appears to be necessary for Taxol-induced apoptosis.

Partial secretome analysis of Caldariomyces fumago reveals extracellular production of the CPO co-substrate H2O2 and provides a coproduction concept for CPO and glucose oxidase.

The culture supernatant of Caldariomyces fumago strains grown in a minimal medium with fructose contains mainly the biotechnologically relevant enzyme chloroperoxidase (CPO) and only minor amounts of other proteins. Our approach to identify the nature of these proteins via peptide mass fingerprinting and transcriptome analysis demonstrated the presence of putative glycosyl hydrolase and glucose oxidase (GOx) enzymes. These activities had been described earlier as parts of the fungus´ halogenation machinery, as they provide CPO with the co-substrate H2O2. The GOx activity was found to have a pH optimum of 5. Compared to the wild type values, GOx activity and glucose-driven MCD chlorination activity in the culture of a white mutant were found to be strongly increased to values of 1-2UmL-1. As most CPO-catalyzed peroxidation reactions also show pH optima at around 5, the C. fumago culture supernatant can provide a highly convenient CPO/GOx source for many reactions with in situ H2O2 production.