Glycopeptide analysis by mass spectrometry may provide an important opportunity in discovery of biomarkers to aid in early detection of Alzheimer's Disease (AD). In this work, we have used a NanoLC-Stepped-HCD-DDA-MS/MS platform and a NanoLC-Stepped-HCD-PRM-MS platform for large-scale screening and quantification of novel N-glycopeptide biomarkers for early detection of AD in patient serum. N-glycopeptides were retrieved from 10 μL of serum in patients with mild cognitive impairment (MCI, a prodromal phase of AD) and normal controls, respectively, after trypsin digestion, glycopeptide enrichment, fractionation, and NanoLC-Stepped-HCD-DDA-MS/MS or NanoLC-Stepped-HCD-PRM-MS analysis. Using a combination of Byonic, Byologic and Skyline softwares, we were able to accomplish both identification and label-free quantitation of site-specific N-glycopeptides between MCI and normal controls. Differential quantitation analysis by Byologic showed that 29 N-glycopeptides derived from 16 glycoproteins were significantly changed in MCI compared to normal controls. Further, HCD-PRM-MS quantitative analysis of the selected N-glycopeptide candidates confirmed that EHEGAIYPDN138TTDFQR_HexNAc(4)Hex(5)-Fuc(2)NeuAc(1) from CERU, and VCQDCPLLAPLN156DTR_HexNAc(4)Hex(5)NeuAc(2) from AHSG can significantly discriminate MCI from normal controls. These two glycopeptides had the area under the receiver operating characteristic curve (AUC) of 0.850 (95% CI, 0.66-1.0) and 0.867 (95% CI, 0.68-1.0), respectively (p<0.05). The result demonstrates that changes in the expression level of the N-glycopeptides provide potential serum biomarkers for detection of AD at a very early stage.
In the latest years spray drying has been adopted from other disciplines as a process to convert liquid proteins/ peptides into more stable dry powder drug substances and products. By removing water, the molecular motion is reduced, which hence can improve storage stability and furthermore remove stringent and costly requirements for the storage temperature. Spray drying offers certain advantages above other drying methods with a more proven track record, amongst others time, cost, scale flexibility, continuous process and particle engineering. There is though still an on going hesitation for the full utilisation of spray drying for manufacturing of dry powder macromolecules in the industry due to the utilisation of heat in the process. For solvent evaporation, input energy is in the form of a hot drying gas around 100-200°C, which in the ears of a peptide/protein specialist may not sound like a recipe for success for such heat labile molecules. This presentation will however go through some common misconceptions about the spray drying process and discuss why spray drying holds a future in securing stable protein/peptide formulations. Furthermore we will present some concrete examples showing that we shouldn’t fear the high temperature used in the process and even in some cases embrace it in order to produce high quality, stable dry powder proteins and peptides.
Salt stress as one of the major abiotic stresses limits the world crop production. Sugar beet monosomic addition line M14 is a unique germplasm with salt stress tolerance, and root is directly exposed to salt stress in the soil. Here we report changes in root membrane proteome of the M14 plants in response to 0, 200 mM and 400 mM NaCl treatment using iTRAQ LC-MS/MS quantitative proteomics. A total of 115 differentially expressed membrane proteins (96 increased and 19 decreased) were identified with a significant fold change of >2.0. The proteins were mainly involved in the processes of transport, signaling, stress and defense, energy, protein degradation, and transcription. Transcriptional changes of 10 genes encoding the differential membrane proteins were analyzed using real-time PCR, and seven genes showed a positive correlation between transcriptional and protein levels. These results have revealed interesting mechanisms underlying the M14 root response to the salt stress, which may have potential applications toward improving crop salt tolerance through genetic engineering and molecular breeding.
This study was conducted to determine the fungal contaminants of garri in Dutsinma metropolis. A total of 150 samples were bought at random, 75 each of white and red garri from three different markets including Wednesday market, Abuja road market and Old market respectively. The fungi isolated using standard microbiological techniques were Alternaria spp. (3.7%), Aspergillus spp. (14.8%), Cladosporium spp. (27.8%), Fusarium spp. (9.3%), Mucor spp. (12.9%), Penicillium spp. (16.7%) and Rhizopus spp. (14.8%) in white garri. For yellow garri: Alternaria spp. (2.9%), Aspergillus spp. (8.8%), Cladosporium spp. (29.4%), Fusarium spp. (8.8%), Mucor spp. (11.8%), Penicillium spp. (20.6%) and Rhizopus spp. (17.6%) with Cladosporium and Rhizopus recording the highest occurrence in both white and yellow garri. Higher fungi species were isolated from white garri (54) compared to yellow (34) samples. Moisture content recorded showed higher content in white garri than in the yellow garri. Identification of the isolated fungi based on their morphological and cultural characteristics was carried out. The results show that consumers are exposed to the risk of aflatoxin poisoning. Efforts should therefore be made to improve the quality of Cassava farina by addressing its handling and processing practices.
Diabetic mellitus and cataracts are common mitochondrial diseases in Indonesia, occurring as a result of a lack of ATP, which is produced in mitochondria via oxidative phosphorylation (OXPHOS). The OXPHOS chain is composed of five complex proteins encoded by genes from nuclear DNA and mitochondrial DNA. One such gene is the CYB gene, with its locus in mitochondrial DNA. The CYB gene encodes part of the complex III subunit, cytochrome b, an electron transfer agent. A novel mutation has been identified at T15458C in type 2 diabetes mellitus patients and T15663C in cataract patients, both of which are in the CYB gene, leading to amino acid alterations S238P and I306T. This study investigated the relationship between T15458C and T15663C mutations in the CYB gene of mitochondrial DNA to the OXPHOS process via an in silico method. Wild type and mutant CYB were modelled via homology modelling using Modeller 9.19 with a 5XTE template, then evaluated using the Ramachandran plot, packaging quality score, and the DOPE profile. The results of the S238P mutant structure analysis showed that proline acts as a helix-breaker due to the loss of a hydrogen bond, while I306T causes a hydrophobic interaction as the helix stabiliser is lost in the mutant. It is assumed the threonine was phosphorylation mutations also destabilises the complex, as proven by the ΔΔG < 0. In conclusion, S238P and I306T mutations affect the function of complex III as an electron transfer agent in the respiratory process.
