Microwave-Assisted Acid and Base Hydrolysis of Intact Proteins Containing Disulfide Bonds for Protein Sequence Analysis by Mass Spectrometry

Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein

Identification of a General O-linked Protein Glycosylation System in Acinetobacter baumannii and Its Role in Virulence and Biofilm Formation

Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening “superbugs” for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics

Profiling of Glycosphingolipids with SCDase Digestion and HPLC-FLD-MS

Lipid components of cells and tissues feature a large diversity of structures that present a challenging problem for molecular analysis. Glycolipids from mammalian cells contain glycosphingolipids (GSLs) as their major glycolipid component, and these structures vary in the identity of the glycan headgroup as well as the structure of the fatty acid and sphingosine (Sph) tails. Analysis of intact GSLs is challenging due to the low abundance of these species. Here, we develop a new strategy for the analysis of lyso-GSL (l-GSL), GSL that retain linkage of the glycan headgroup with the Sph base. The analysis begins with digestion of a GSL sample with sphingolipid ceramide N-deacylase (SCDase), followed by labelling with an amine-reactive fluorophore. The sample was then analyzed by HPLC-FLD-MS and quantitated by addition of an external standard. This method was compared analysis of GSL glycans after cleavage by an Endoglycoceramidase (EGCase) enzyme and labeling with a fluorophore (2-anthranilic acid, 2AA). The two methods are complementary, with EGCase providing improved signal (due to fewer species) and SCDase providing analysis of lyso-GSL. Importantly the SCDase method provides Sph composition of GSL species. We demonstrate the method on cultured human cells (Jurkat T cells) and tissue homogenate (porcine brain)

Persistent reduction in sialylation of cerebral glycoproteins following postnatal inflammatory exposure

Abstract Background The extension of sepsis encompassing the preterm newborn’s brain is often overlooked due to technical challenges in this highly vulnerable population, yet it leads to substantial long-term neurodevelopmental disabilities. In this study, we demonstrate how neonatal neuroinflammation following postnatal E. coli lipopolysaccharide (LPS) exposure in rat pups results in persistent reduction in sialylation of cerebral glycoproteins. Methods Male Sprague-Dawley rat pups at postnatal day 3 (P3) were injected in the corpus callosum with saline or LPS. Twenty-four hours (P4) or 21 days (P24) following injection, brains were extracted and analyzed for neuraminidase activity and expression as well as for sialylation of cerebral glycoproteins and glycolipids. Results At both P4 and P24, we detected a significant increase of the acidic neuraminidase activity in LPS-exposed rats. It correlated with significantly increased neuraminidase 1 (Neu1) mRNA in LPS-treated brains at P4 and with neuraminidases 1 and 4 at P24 suggesting that these enzymes were responsible for the rise of neuraminidase activity. At both P4 and P24, sialylation of N-glycans on brain glycoproteins decreased according to both mass-spectrometry analysis and lectin blotting, but the ganglioside composition remained intact. Finally, at P24, analysis of brain tissues by immunohistochemistry showed that neurons in the upper layers (II–III) of somatosensory cortex had a reduced surface content of polysialic acid. Conclusions Together, our data demonstrate that neonatal LPS exposure results in specific and sustained induction of Neu1 and Neu4, causing long-lasting negative changes in sialylation of glycoproteins on brain cells. Considering the important roles played by sialoglycoproteins in CNS function, we speculate that observed re-programming of the brain sialome constitutes an important part of pathophysiological consequences in perinatal infectious exposure

Characterization of <i>A. baumannii</i> ATCC 17978 pathogenesis in a murine septicemia model.

<p>A) Determination of the LD₅₀ of <i>A. baumannii</i> ATCC 17978. Groups of 5 mice were injected with serial dilutions of <i>A. baumannii</i> WT to determine the LD₅₀ which was calculated to be 6.49×10⁴ CFU @ 18 hrs post infection. B) Murine competition septicemia between <i>A. baumannii</i> WT and <i>ΔpglL</i>. Groups of 3 mice were injected with ∼1∶1 WT to Δ<i>pglL</i> CFU's and were sacrificed after 18 hrs, spleens were harvested, and bacterial load determined.</p

Identification of seven <i>O-</i>glycosylated proteins with nine unique glycosylation sites by ZIC HILIC enrichment of tryptically digested <i>A. baumannii</i> ATCC 17978 membrane extracts.

<p>Identification of seven <i>O-</i>glycosylated proteins with nine unique glycosylation sites by ZIC HILIC enrichment of tryptically digested <i>A. baumannii</i> ATCC 17978 membrane extracts.</p

<i>A. baumannii</i> requires PglL_Ab for biofilm formation.

<p>A) Quantitative biofilm formation on polystyrene 96 well plates by strains incubated without perturbation in LB at 30°C. The bars indicate the means for 8 replicates. The error bars indicate the standard deviation of the means. Asterisks indicate significant differences (*, <i>P</i><0.005 [<i>t</i> test; <i>n</i> = 8]; **, <i>P</i><0.001 [<i>t</i> test; <i>n</i> = 8]). B) The median surface coverage after incubation for 2 h in flow cell chambers of the WT, <i>ΔpglL</i>, <i>ΔpglL</i> pWH1266 and <i>ΔpglL</i> ppglL was determined by the COMSTAT software. For each strain at least six micrographs from three independent experiments were analyzed. The error bars indicate the interquartile range. Asterisks indicate significant differences (*, <i>P</i><0.05 [Mann-Whitney U test; <i>n</i> = 6]). C)–E) Image stacks of the WT, <i>ΔpglL</i>, <i>ΔpglL</i> pWH1266 and <i>ΔpglL</i> ppglL biofilms grown in flow cells for 24 h were analyzed for the biomass as well as the maximum and average thickness using the COMSTAT software. Shown are the medians of at least six image stacks from three independent experiments for each strain. The error bars indicate the interquartile range. Asterisks indicate significant differences (*, <i>P</i><0.05 [Mann-Whitney U test; <i>n</i> = 6]). F) Shown are representative confocal laser scanning microscopy images of the WT (upper row) and <i>ΔpglL</i> mutant (lower row) biofilms grown in flow cells for 24 h. The first three images represent horizontal (xy, large panel) and vertical (xz and yz, side panels) projections at different z-levels (from left to right 0.2 µm, 3 µm and 6 µm). The fourth micrograph of each row represents a three-dimensional image analyzed by the AMIRA software package of the WT and <i>ΔpglL</i> mutant biofilms, respectively.</p

Identification of additional glycoproteins in <i>A. baumannii</i> ATCC 17978.

<p>Tryptically digested membranes were enriched via ZIC-HILIC and analyzed by LC-MS and HCD MS-MS. All spectra were analyzed for the diagnostic oxonium ion of 301.10 m/z, and positive spectra were analyzed manually to identify the glycopeptide. This spectra is representative of each glycopeptide identified in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002758#ppat-1002758-t002" target="_blank">Table 2</a>. A) ITMS-CID of the precursor ion at <i>m/z</i> 1999.943 reveals the pentasaccharide attached to the peptide <b>AKPASTPAVK</b>. B) FTMS-HCD of the precursor ion at <i>m/z</i> 1999.943 reveals the peptide sequence <b>AKPASTPAVK</b>.</p

Comparison of <i>A. baumannii</i> WT and Δ<i>pglL</i> membrane extracts by 2D-DIGE.

<p>Analysis of the membrane proteome of <i>A. baumannii</i> WT strain (A), Δ<i>pglL</i> strain (B), and merge (C). Spots WT1 and WT2 only present in the WT strain (green) whereas MT1 and MT2 were only present in the Δ<i>pglL</i> strain (red). MALDI-TOF MS analysis identified WT1 and MT1 spots as A1S_3626 protein and WT2 and MT2 spots as A1S_3744 protein.</p