Analysis of MALDI-TOF mass spectrometry data for discovery of peptide and glycan biomarkers of hepatocellular carcinoma

This paper presents computational methods to analyze MALDI-TOF mass spectrometry data for quantitative comparison of peptides and glycans in serum. The methods are applied to identify candidate biomarkers in serum samples of 203 participants from Egypt; 73 hepatocellular carcinoma (HCC) cases, 52 patients with chronic liver disease (CLD) consisting of cirrhosis and fibrosis cases, and 78 population controls. Two complementary sample preparation methods were applied prior to generating mass spectra: (1) low molecular weight (LMW) enrichment of each serum sample was carried out for MALDI-TOF quantification of peptides, and (2) glycans were enzymatically released from proteins in each serum sample and permethylated for MALDI-TOF quantification of glycans. A peak selection algorithm was applied to identify the most useful peptide and glycan peaks for accurate detection of HCC cases from high-risk population of patients with CLD. In addition to global peaks selected by the whole population based approach, where identically labeled patients are treated as a single group, subgroup-specific peaks were identified by searching for peaks that are differentially abundant in a subgroup of patients only. The peak selection process was preceded by peak screening, where we eliminated peaks that have significant association with covariates such as age, gender, and viral infection based on the peptide and glycan spectra from population controls. The performance of the selected peptide and glycan peaks was evaluated in terms of their ability in detecting HCC cases from patients with CLD in a blinded validation set and through the cross-validation method. Finally, we investigated the possibility of using both peptides and glycans in a panel to enhance the diagnostic capability of these candidate markers. Further evaluation is needed to examine the potential clinical utility of the candidate peptide and glycan markers identified in this study.     

Structure–function analysis of hepatitis C virus envelope glycoproteins E1 and E2

Hepatitis C virus (HCV) is the leading cause of chronic liver disease in humans. The envelope proteins of HCV are potential candidates for vaccine development. The absence of three-dimensional (3D) structures for the functional domain of HCV envelope proteins [E1.E2] monomer complex has hindered overall understanding of the virus infection, and also structure-based drug design initiatives. In this study, we report a 3D model containing both E1 and E2 proteins of HCV using the recently published structure of the core domain of HCV E2 and the functional part of E1, and investigate immunogenic implications of the model. HCV [E1.E2] molecule is modeled by using aa205–319 of E1 to aa421–716 of E2. Published experimental data were used to further refine the [E1.E2] model. Based on the model, we predict 77 exposed residues and several antigenic sites within the [E1.E2] that could serve as vaccine epitopes. This study identifies eight peptides which have antigenic propensity and have two or more sequentially exposed amino acids and 12 singular sites are under negative selection pressure that can serve as vaccine or therapeutic targets. Our special interest is ₂₈₅FLVGQLFTFSPRRHW₂₉₉ which has five negatively selected sites (L286, V287, G288, T292, and G303) with three of them sequential and four amino acids exposed (F285, L286, T292, and R296). This peptide in the E1 protein maps to dengue envelope vaccine target identified previously by our group. Our model provides for the first time an overall view of both the HCV envelope proteins thereby allowing researchers explore structure-based drug design approaches.     

Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma

A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p < 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.     

The pollutants in rime and fog water and in air at Milesovka Observatory (Czech Republic)

Samples of rime/fog water and dust in the air were collected in order to compare concentrations of pollutants. Particular attention was paid to particles of heavy metals (Al, As, Cu, Fe, Pb, Ti and Zn). The concentrations of pollutants from the air are different in rime water and fog water. Both (fog) water and ice crystals fixed Ti, Cu, As and Pb ions minimally (less than 1%). Rime captured 11.6% Al, 9.3% Zn, 4.4% Fe and 91.2% Mn from the air. Fog water absorbed 9.8% Al, 9.0% Fe, 55.6% Mn, and 48.7% Zn from the air. Fog water absorbed Zn much better (48.7%) than rime (9.3%). Rime absorbed Mn better (91.2%) than fog water (55.6%).     

Proteome-wide analysis of single-nucleotide variations in the N-glycosylation sequon of human genes

N-linked glycosylation is one of the most frequent post-translational modifications of proteins with a profound impact on their biological function. Besides other functions, N-linked glycosylation assists in protein folding, determines protein orientation at the cell surface, or protects proteins from proteases. The N-linked glycans attach to asparagines in the sequence context Asn-X-Ser/Thr, where X is any amino acid except proline. Any variation (e.g. non-synonymous single nucleotide polymorphism or mutation) that abolishes the N-glycosylation sequence motif will lead to the loss of a glycosylation site. On the other hand, variations causing a substitution that creates a new N-glycosylation sequence motif can result in the gain of glycosylation. Although the general importance of glycosylation is well known and acknowledged, the effect of variation on the actual glycoproteome of an organism is still mostly unknown. In this study, we focus on a comprehensive analysis of non-synonymous single nucleotide variations (nsSNV) that lead to either loss or gain of the N-glycosylation motif. We find that 1091 proteins have modified N-glycosylation sequons due to nsSNVs in the genome. Based on analysis of proteins that have a solved 3D structure at the site of variation, we find that 48% of the variations that lead to changes in glycosylation sites occur at the loop and bend regions of the proteins. Pathway and function enrichment analysis show that a significant number of proteins that gained or lost the glycosylation motif are involved in kinase activity, immune response, and blood coagulation. A structure-function analysis of a blood coagulation protein, antithrombin III and a protease, cathepsin D, showcases how a comprehensive study followed by structural analysis can help better understand the functional impact of the nsSNVs.     

Biochemical studies on the production of neuraminidase by environmental isolates of Vibrio cholerae non-O1 from Bulgaria

Neuraminidase is a key factor in the infectious process of many viruses and pathogenic bacteria. The neuraminidase enzyme secreted by the etiological agent of cholera?- Vibrio cholerae О1?- is well studied in contrast with the one produced by non-O1/non-O139 V.?cholerae. Environmental non-O1/non-O139 V. cholerae isolates from Bulgaria were screened for production of neuraminidase. The presence of the neuraminidase gene nanH was detected in 18.5% of the strains. Тhe strain showing highest activity (30 U/mL),?V. cholerae non-O1/13, was used to investigate the enzyme production in several media and at different aeration conditions. The highest production of extracellular neuraminidase was observed under microaerophilic conditions, which is possibly related to its role in the infection of intestine epithelium, where the oxygen content is low. On the other hand, this is another advantage of the microbe in such microaerophilic environments as sediments and lake mud. The highest production of intracellular neuraminidase was observed at anaerobic conditions. The ratio of extracellular to intracellular neuraminidase production in V. cholerae was investigated. The temperature optimum of the enzyme was determined to be 50?°C and the pH optimum to be?5.6-5.8.