ABSTRACT: Mass screening of crude extracts prepared from Actinokineospora auranticolor fermentation and LC-MSMS data acquisition for structure analysis.
Project description:This project aims to analyse the proteins of outer membrane vesicles (OMVs) released by a Serratia bacteria strain Su_YN1, which is isolated from field-caught mosquito gut. Su_YN1 produces massive OMVs under host serum induction. The data contain LC-MSMS results of the OMVs released without serum induction (WT-), and serum induced OMVs of WT strain (WT+) and several gene disruption strains (disruption of the ABC transporter genes ABC1 and ABC2). This project was conducted by CEMPS lab and Shanghai hoogen biotech co.ltd. The CEMPS lab prepared the purified OMV samples and Shanghai hoogen biotech co.ltd conducted the LC-MSMS analysis.
Project description:BACKGROUND:The presence of a wide range of bioactive organic pollutants in wastewater and municipal water sources is raising concerns about their potential effects on humans. Not surprisingly, various approaches are being explored that can efficiently degrade these persistent organic pollutants. Use of peroxidases has recently been recognized as a novel remediation approach that may have potential advantages over conventional degradation techniques. However, testing the abilities of different peroxidases to degrade diverse emerging pollutants is tedious and cumbersome. RESULTS:In the present study, we present a rapid and robust approach to easily test the degradability of 21 different emerging pollutants by five different peroxidases (soybean peroxidase, chloroperoxidase, lactoperoxidase, manganese peroxidase, and horseradish peroxidase) using an LC-MSMS approach. Furthermore, this approach was also used to examine the role of a redox mediator in these enzymatic degradation assays. Our results show that some of the organic pollutants can be easily degraded by all five of the peroxidases tested, whereas others are only degraded by a specific peroxidase (or when a redox mediator was present) and there are some that are completely resistant to degradation by any of the peroxidases tested (even in the presence of a redox mediator). The degradation of furosemide and trimethoprim by soybean peroxidase and chloroperoxidase, respectively, was investigated in detail by examining the transformation products generated during their degradation. Some of the products generated during enzymatic breakdown of these pollutants have been previously reported by others, however, we report many new transformation products. CONCLUSIONS:LC-MSMS approaches, like the one described here, can be used to rapidly evaluate the potential of different peroxidases (and redox requirements) to be used as bioremediation agents. Our preliminary result shows peroxidases hold tremendous potential for being used in a final wastewater treatment step.
Project description:We constructed genetically modified Eimeria acervulina that expressed VP2 protein, a protective antigen from infectious bursal disease virus (IBDV), on the surface or in the microneme of sporozoites. we identified the transgenic E. acervulina expressing VP2 by RT-qPCR,LC-MSMS and Werstern Blot.
Project description:Stable isotope coding technique in combination with mass spectrometry has emerged as a powerful tool to accurately identify and differentially quantify proteins within complex protein mixtures. We present a novel methodology to increase the yield of quantified proteins while maintaining a high stable-isotopic labeling efficacy. With this approach, intact proteins in complex biological sample such as sera are labeled with the designated dual stable isotope coding (DSIC) systems. In brief, intact proteins are coded sequentially with acrylamide to label Cysteine residues (Cys) and with succinic anhydride to label Lysine residues (Lys). Protein samples coded with this dual stable isotope are subjected to an online 2D-HPLC fractionation. The resolved protein fractions are individually digested with trypsin and analyzed with nano LC-MS/MSMS. Our results show that the DSIC labeling efficiency is 100% for Cysteine (Cys) labeled with acrylamide and 98% for Lysine (Lys) labeled with succinic anhydride. A comparative analysis of DSIC labeling and single labeling of Cysteine residues was made. Analysis of an entire anion-exchange chromatography subfraction of sera yielded 165 identified proteins (criteria: error rate <5% and unique peptides >or=2), 104 of which were quantified using the single labeling method (i.e., Cysteine acrylamide labeling only). In contrast, using same criteria for identification, a total 185 proteins were identified and 174 proteins were quantified using the DSIC labeling technique.
Project description:With the optional setting of multiple stepped collisional energies (NCEs), higher-energy collisional dissociation (HCD) as available on Orbitrap instruments is a widely adopted dissociation method for intact N-glycopeptide characterization, where peptide backbones and N-glycan moieties are selectively fragmented at high and low NCEs, respectively. Initially, a dependent setting of a central value plus minus a variation is available to the users to set up NCEs, and the combination of 30±10% to give the energies 20%/30%/40% has been mostly adopted in the literature. With the recent availability of independent NCE setup, we found that the combination of 20%/30%/30% is better than 20%/30%/40%; in the analysis of complex intact N-glycopeptides enriched from gastric cancer tissues, total IDs with spectrum-level FDR≤1%, site-specific IDs with site-determining fragment ions and structure-specific IDs with structure-diagnostic fragment ions were increased by 42% (4,767->6,746), 57% (599->942), and 97% (1771->3495), respectively. This finding will benefit all the coming N-glycoproteomics studies using HCD as the dissociation method.
Project description:Enzymatic Methyl-seq (EM-seq) is an enzyme-based method giving us reliable methylome data from small amounts of purified DNA. However, it is unclear whether EM-seq library can be constructed from crude cell lysate containing genomic DNA. We evaluated the quality of EM-seq libraries directly prepared from crude cell lysate.
Project description:This article contains the peptide data obtained while performing disulfide bond mapping of the recombinant Plasmodium falciparum protein, Pfs25, produced from the baculovirus expression system. Pfs25 is a malaria transmission-blocking vaccine candidate, with a compact and complex structure including 22 cysteines. This supplementary data is related to the research "Disulfide bond mapping of Pfs25, a recombinant malaria transmission blocking vaccine candidate" (Lee et al., 2018) [1]. In brief, Pfs25 was digested with trypsin/Lys-C and derived peptides separated by High Performance Liquid Chromatography (HPLC) and analyzed by mass spectrometry (MS) by MSE fragmentation. The theoretical peptides and their respective masses along with disulfide bond locations with linked peptides are presented here alongside the mass spectrometry analysis. The raw mass spectrometry data is made available through the Mass Spectrometry Interactive Virtual Environment (MassIVE) with identifier: MSV000081982.
Project description:The evaluation of the protein glycosylation states of samples of aflibercept obtained from three different regions was conducted by site-specific N-linked glycan microheterogeneity profiling. Glycopeptide-based nano-LC MSMS mapping of tryptic-digested samples of each aflibercept lot provided site-specific information about glycan microheterogeneity on each of the five N-glycosylation sites (two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region). Next, the glycopeptide-mapping results obtained from the three different aflibercept lots were compared to evaluate the similarity between the samples. Three aflibercept lots showed a high degree of similarity in glycan composition, fucosylation level, sialylation level, and branching, when all five N-glycosylation sites were assessed together as a group. On the other hand, noticeable variations between lots in the glycan types and sialylation levels on the two sites of the VEGFR-2 domain were observed when each of the five N-glycosylation sites were assessed using the glycopeptide-based method. The presence of N-glycolylneuraminic acid (NeuGc) glycans, which may mediate adverse immune reactions in antibody therapeutics, were also detected on the sites of VEGFR1 and VEGFR2 domains, but not on the IgG Fc domain site. These results imply that analyses of the glycosylation profiles of fusion proteins containing multiple N-glycosylation sites, such as aflibercept, being done as a part of quality control for the therapeutics manufacturing process or for biosimilar development, can be done with a more applicable outcome by assessing each site separately.
Project description:IntroductionScreening for chronic kidney disease relies on accurate and precise creatinine measurements. Traditionally, creatinine is measured in serum or plasma using high-throughput chemistry analyzers. However, dried blood spots (DBS) can also be utilized to improve testing access.MethodsSamples were obtained from a 6 mm DBS punch, which was reconstituted in water before undergoing an acetonitrile crash. The resulting supernatant was diluted using an 80:20 acetonitrile: water before injection. Creatinine was identified using an isocratic gradient, and detected using an API 4000 triple quadrupole mass analyzer. Quantification relied on matrix-matched calibrators, with values harmonized to the Roche Cobas enzymatic assay. Validation studies assessing method performance included precision, linearity, accuracy, method comparison, stability, interference, and matrix effects.ResultsThe LC-MSMS assay was linear from 0.3 to 20 mg/dL (y = 1.02x-0.11; R2 = 0.996). Precision ranged from 5.2 to 8.1 % using matrix-matched controls (n = 4) that spanned the analytical measurement range. LC-MSMS results corresponded to the enzymatic assay (Roche) with a fitted line equation of y = 0.956x-0.07 (R2 = 0.995; n = 173). The Siemens and Roche enzymatic assays demonstrated higher accuracy in correlating to the DBS creatinine concentration (n = 40 paired venous/DBS collections) compared to the Beckman Jaffe assay (-2.5 % and -0.8 % versus -6.3 % and -4.1 %, respectively) or the iSTAT (-28.4 % and -27.1 %, respectively). Accuracy was unaffected by hematocrit, blood spot volume, excess IgG or IgA, or hypertriglyceridemia. No matrix effects were observed, and both extraction and processing efficiency were robust.Ambient stability extended to at least 10 days, and exposure to extreme temperature did not affect the creatinine results.ConclusionWe successfully developed an accurate and precise LC-MSMS method for quantifying creatinine in DBS.