On-MALDI-Target N-Glycan Nonreductive Amination by 2-Aminobenzoic Acid.

2-Aminobenzoic acid (2-AA) is widely used as a labeling reagent to derivatize released N-glycans at their free reducing terminus by reductive amination. 2-AA-labeled glycans have increased mass spectrometric sensitivity for their identification and enable fluorescence-chromatography-based glycan quantification. Drawbacks are that the labeling process is labor intensive and time consuming. Clean up of labeled glycans via removal of excess of labeling reagents often leads to sample losses. Here, we report use of 2-AA for labeling N-glycans on a MALDI target through nonreductive amination, while simultaneously functioning as a matrix in MALDI-MS glycan analysis. Coupling 2-AA to glycans results in significant increases of glycan anionic signals as compared to that using the traditional 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. The on-MALDI-target sample preparation is a single-step protocol with high derivatization efficiency. It is also noticed that 2-AA-labeled glycan generated dominant deprotonated molecular anions with much fewer and low-intensity sodium adducts and therefore greatly simplified glycan profiles. We further explored its application in the N-glycan profile of a biotherapeutic monoclonal antibody and was able to achieve sensitive glycan identification at a low microgram level of glycoprotein. This 2-AA on-MALDI-target glycan derivatization eliminates tedious sample preparation and avoids sample loss. It is generally applicable for other applications (e.g., glycomics), where limited amounts of glycoproteins are available for analysis.

Defining Changes in the Spatial Distribution and Composition of Brain Lipids in the Shiverer and Cuprizone Mouse Models of Myelin Disease.

Myelin is composed primarily of lipids and diseases affecting myelin are associated with alterations in its lipid composition. However, correlation of the spatial (in situ) distribution of lipids with the disease-associated compositional and morphological changes is not well defined. Herein we applied high resolution matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), immunohistochemistry (IHC), and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) to evaluate brain lipid alterations in the dysmyelinating shiverer (Shi) mouse and cuprizone (Cz) mouse model of reversible demyelination. MALDI-IMS revealed a decrease in the spatial distribution of sulfatide (SHexCer) species, SHexCer (d42:2), and a phosphatidylcholine (PC) species, PC (36:1), in white matter regions like corpus callosum (CC) both in the Shi mouse and Cz mouse model. Changes in these lipid species were restored albeit not entirely upon spontaneous remyelination after demyelination in the Cz mouse model. Lipid distribution changes correlated with the local morphological changes as confirmed by IHC. LC-ESI-MS analyses of CC extracts confirmed the MALDI-IMS derived reductions in SHexCer and PC species. These findings highlight the role of SHexCer and PC in preserving the normal myelin architecture and our experimental approaches provide a morphological basis to define lipid abnormalities relevant to myelin diseases.

Endovascular repair with chimney technique for juxtarenal aortic aneurysm: a single center experience.

OBJECTIVE: Chimney endovascular aneurysm repair (Ch-EVAR) is a cheap and immediately available method for treatment of juxtarenal aortic aneurysms (JRAAs). The aim of this study was to report experiences and results with balloon expandable stent (BES) for Ch-EVAR. METHODS: From January 2008 to June 2013 (mean time, 26 ± 13 months), 42 patients who underwent Ch-EVAR were retrospectively reviewed. All patients were thought to be at high risk for open surgery and were unsuitable (because of financial issues and fear of delays) for fenestrated endovascular aneurysm repair (FEVAR). RESULTS: 42 patients (35 males; mean age 71 ± 7 years) underwent chimney procedures for 56 renal arteries with 70 BESs (59 hippocampus and 11 genesis). Median follow up was 26 months (range 6-64 months). Primary technical success was 100%. Thirty day mortality was 0%. Completion angiography showed that eight cases (19%) had a type I endoleak. The 6 month follow up CT scans demonstrated three cases with complete regression of endoleak, two cases without aneurysmal growth, and three cases with an increase in aneurysmal diameter of less than 10 mm. Three patients had contrast induced nephropathy (CIN) after Ch-EVAR, two of whom had acute renal failure (ARF) and continue to require hemodialysis. Two deaths occurred during follow up, both unrelated to the aorta: one with a hemispheric stroke at 6 months and one with respiratory failure at 12 months. Therefore, the overall follow up mortality was 5%. Occlusion of one chimney stent occurred 3 months after the procedure, meaning renal artery patency rate was 98%. The aneurysm diameter reduced from 74 ± 9 mm to 64 ± 10 mm during follow up (p < .05). CONCLUSION: Ch-EVAR can be used to treat JRAAs with suitable anatomical conditions. However, complications of type I endoleak were not uncommon, and, therefore, further studies are required to prove its efficiency for JRAAs.

CPEB1, a histone-modified hypomethylated gene, is regulated by miR-101 and involved in cell senescence in glioma.

Epigenetic mechanisms have important roles in carcinogenesis. We certified that the mRNA translation-related gene cytoplasmic polyadenylation element-binding protein 1 (CPEB1) is hypomethylated and overexpressed in glioma cells and tissues. The knockdown of CPEB1 reduced cell senescence by regulating the expression or distribution of p53 in glioma cells. CPEB1 is also regulated directly by the tumor suppressor miR-101, a potential marker of glioma. It is known that the histone methyltransferase enhancer of zeste homolog 2 (EZH2) and embryonic ectoderm development (EED) are direct targets of miR-101. We demonstrated that miR-101 downregulated the expression of CPEB1 through reversing the methylation status of the CPEB1 promoter by regulating the presence on the promoter of the methylation-related histones H3K4me2, H3K27me3, H3K9me3 and H4K20me3. The epigenetic regulation of H3K27me3 on CPEB1 promoter is mediated by EZH2 and EED. EZH2 has a role in the regulation of H3K4me2. Furthermore, the downregulation of CPEB1 induced senescence in a p53-dependent manner.

Discovery and investigation of O-xylosylation in engineered proteins containing a (GGGGS)n linker.

Protein engineering is a powerful tool for designing or modifying therapeutic proteins for enhanced efficacy, greater safety, reduced immunogenicity, and better delivery. GGGGS [(G4S)n] linkers are commonly used when engineering a protein, because of their flexibility and resistance to proteases. However, post-translational modifications (PTMs) can occur at the Ser residue in these linkers. Here, we report, for the first time, the occurrence of O-xylosylation at the serine residue in (G4S)n>2 linkers. The O-xylosylation was discovered as a result of molecular mass determination, peptide mapping analysis, and MS/MS sequencing. Our investigation showed that (i) O-xylosylation is a common PTM for (G4S)(n>2) linkers; (ii) GSG is the motif for O-xylosylation; and (iii) the total amount of xylosylation per linker increases as the number of GSG motifs in the linker increases. Our investigation has also shown that the O-xylosylation level is clone-dependent, to a certain degree, but the xylosylation level varies considerably among the proteins examined-from <2% to >25% per linker-likely depending on the accessibility to the sites by the xylosyltransferase. Our work demonstrates that potential therapeutic proteins containing (G4S)n linkers should be closely monitored for O-xylosylation in order to ensure that drugs are homogeneous and of high quality. The strategies for elimination and reduction of O-xylosylation were also examined and are discussed.

Purification and characterization of a silica-induced bronchoalveolar lavage protein with fibroblast growth-promoting activity.

Experimentally induced silicosis provides a good model for chronic interstitial pulmonary inflammation and fibrosis. In the present study, a specific single polypeptide with an apparent molecular mass of 58,000 and a pl of 4.5 was purified and characterized from the bronchoalveolar lavage fluid of silicotic rats. The same protein was also isolated from both the extract and conditioned medium of alveolar macrophages of silicotic rats. Therefore, this protein was termed an inducible silicotic (rat) bronchoalveolar lavage protein-p58 (iSBLP58) or an inducible silicotic (rat) pulmonary macrophage factor (iSPMF-p58). iSBLP58 has been purified to homogeneity by a combination of gel permeation, Mono Q ion exchange, and reverse-phase high performance liquid chromatography. This polypeptide displayed a potent fibroblast growth-promoting activity in vitro. The sequence of the first 15 NH2-terminal amino acids was determined and was found to have high sequence homology with members of the mammalian chitinase-like protein family, which includes human cartilage gp39, mammalian oviduct-specific glycoprotein, and a secretory protein from activated mouse macrophages.