14 research outputs found
Proteomics Analyses of the Opportunistic Pathogen Burkholderia vietnamiensis Using Protein Fractionations and Mass Spectrometry
The main objectives of this work were to obtain a more extensive coverage of the Burkholderia vietnamiensis proteome than previously reported and to identify virulence factors using tandem mass spectrometry. The proteome of B. vietnamiensis was precipitated into four fractions to as extracellular, intracellular, cell surface and cell wall proteins. Two different approaches were used to analyze the proteins. The first was a gel-based method where 1D SDS-PAGE was used for separation of the proteins prior to reverse phase liquid chromatography tandem mass spectrometry (LC-MS/MS). The second method used MudPIT analysis (Multi dimensional Protein Identification Technique), where proteins are digested and separated using cation exchange and reversed phase separations before the MS/MS analysis (LC/LC-MS/MS). Overall, gel-based LC-MS/MS analysis resulted in more protein identifications than the MudPIT analysis. Combination of the results lead to identification of more than 1200 proteins, approximately 16% of the proteins coded from the annotated genome of Burkholderia species. Several virulence factors were detected including flagellin, porin, peroxiredoxin and zinc proteases
Formation of peptide aggregates during ESI: size, charge, composition, and contributions to noise
AbstractIon mobility/time-of-flight techniques have been used to examine the onset of aggregation in model systems of Gly-Xxx (where Xxxx = Ala, Asn, Asp, Gln, Glu, His, Leu, Ser, Thr, and Trp) dipeptides. Under the experimental conditions employed, there is evidence that simple binary and quaternary mixtures of these dipeptides produce clusters containing as many as 16 to 75 peptide units (and 1 to 7 charges). In some systems, cluster compositions appear to come about largely from statistical association of peptide units; other dipeptide mixtures (and generally for small clusters) show evidence for nonstatistical behavior which could arise from some differences in gas-phase or solution thermochemistry. The minimum aggregate size appears to be largely determined by the charge state. Average thresholds for aggregate formation in the z = 2, 3, and 4 charge state families occur at m/z ā¼500, 660, and 875, respectively. We briefly consider the idea that aggregates formed during electrospray ionization (ESI) may contribute to the background signal observed in the analysis of complex peptide mixtures
IRMPD spectroscopy shows that AGG forms an oxazolone b2+ ion.
International audienceInfrared multiple photon dissociation (IRMPD) spectroscopy combined with theoretical vibrational spectra provides a powerful tool for probing structure. This technique has been used to probe the structure of protonated cyclic AG and the b(2)(+) ion from AGG. The experimental spectrum for protonated cyclo AG compares very well with the theoretical spectra for a diketopiperazine. The spectrum corresponds best to a combination of two structures protonation at the alanine and glycine amide oxygens. The experimental spectrum for the b(2)(+) ion from protonated AGG matches best to the theoretical spectrum for an oxazolone structure protonated on the ring nitrogen. In particular, the carbonyl stretching band at 1970 cm(-1) is blue-shifted by approximately 200 cm(-1) compared to the experimental spectrum for protonated cAG, indicating that these two structures are distinct. This is the first time that an IRPD spectrum of a b(2)(+) ion has been obtained and, for this ion, the oxazolone structure proposed based on prior calculations and experiments is confirmed by the spectroscopic method
Automated Affinity Capture and On-Tip Digestion to Accurately Quantitate <i>in Vivo</i> Deamidation of Therapeutic Antibodies
Deamidation
of therapeutic antibodies may result in decreased drug
activity and undesirable changes in pharmacokinetics and immunogenicity.
Therefore, it is necessary to monitor the deamidation levels [during
storage] and after <i>in vivo</i> administration. Because
of the complexity of <i>in vivo</i> samples, immuno-affinity
capture is widely used for specific enrichment of the target antibody
prior to LCāMS. However, the conventional use of bead-based
methods requires large sample volumes and extensive processing steps.
Furthermore, with automation difficulties and extended sample preparation
time, bead-based approaches may increase artificial deamidation. To
overcome these challenges, we developed an automated platform to perform
tip-based affinity capture of antibodies from complex matrixes with
rapid digestion and peptide elution into 96-well microtiter plates
followed by LCāMS analysis. Detailed analyses showed that the
new method presents high repeatability and reproducibility with both
intra and inter assay CVs < 8%. Using the automated platform, we
successfully quantified the levels of deamidation of a humanized monoclonal
antibody in cynomolgus monkeys over a time period of 12 weeks after
administration. Moreover, we found that deamidation kinetics between <i>in vivo</i> samples and samples stressed <i>in vitro</i> at neutral pH were consistent, suggesting that the <i>in vitro</i> stress test may be used as a method to predict the liability to
deamidation of therapeutic antibodies <i>in vivo</i>
Citizen science in schools: Engaging students in research on urban habitat for pollinators
Citizen science can play an important role in school science education. Citizen science is particularly relevant to addressing current societal environmental sustainability challenges, as it engages the students directly with environmental science and gives students an understanding of the scientific process. In addition, it allows students to observe local representations of global challenges. Here, we report a citizen science programme designed to engage school-age children in real-world scientific research. The programme used standardized methods deployed across multiple schools through scientistāschool partnerships to engage students with an important conservation problem: habitat for pollinator insects in urban environments. Citizen science programmes such as the programme presented here can be used to enhance scientific literacy and skills. Provided key challenges to maintain data quality are met, this approach is a powerful way to contribute valuable citizen science data for understudied, but ecologically important study systems, particularly in urban environments across broad geographical areas