Glycan-specific precipitation of glycopeptides in high organic content sample solvents used in HILIC.
Ontology highlight
ABSTRACT:
SUBMITTER: Kozlik P
PROVIDER: S-EPMC7305521 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Elevated serum prostate-specific antigen (PSA) levels in body fluids may indicate prostate cancer (PCa), but it is noted that the clinical performance is rather poor. Specificity and sensitivity values of 20 and 94% at a cutoff value of 4.1 ng/mL, respectively, result in overdiagnosis and unnecessary interventions. Previous exploratory studies have indicated that the glycosylation of PSA potentially leads to improved PCa diagnosis based on qualitative analyses. However, the applied methods are not suited for a quantitative evaluation or implementation in a medical laboratory. Therefore, in this proof-of-principle study, we have evaluated the use of hydrophilic interaction liquid chromatography (HILIC) in combination with targeted quantitative mass spectrometry for the sialic acid linkage-specific analysis of PSA glyco-proteoforms based on either trypsin or ArgC peptides. The efficiency of PSA proteolysis was optimized as well as the glycopeptide separation conditions (buffer type, strength, and pH). The HILIC-based analysis of PSA glyco-proteoforms presented here has the potential for the clinical validation of patient cohorts. The method shows the feasibility of the use of a HILIC stationary phase for the separation of isomeric glycopeptides to detect specific glyco-proteoforms. This is the first step toward the development and evaluation of PSA glyco-proteoforms for use in a clinical chemistry setting aiming for improved PCa diagnosis or screening.
| S-EPMC8280738 | biostudies-literature
Project description:Typing of amyloidosis by mass spectrometry (MS) based proteomic analysis contribute to the diagnosis of amyloidosis. For MS analysis, laser microdissection (LMD) is used for amyloid specific sampling. This study aimed to establish a method for selectively extracting amyloids from formalin-fixed, paraffin-embedded (FFPE) specimens by organic solvent instead of LMD. The extracts using dimethyl sulfoxide (DMSO), dimethylformamide (DMF), methanol, trifluoroethanol (TFE) or hexafluoro-2-propanol from FFPE brain of alzheimer's disease mouse model generated protein bands on SDS-PAGE, and Aβ was identified in the extract of DMF using mass spectrometry. The extract using DMSO from the kidney of a AA amyloidosis patient produced a protein band in SDS-PAGE. This protein band was identified to be serum amyloid A (SAA) by Western blotting and mass spectrometry. Circular dichroism spectrometry revealed that the secondary structures of Aβ and transthyretin were converted to α-helices from β-sheets in TFE. Our results suggest that organic solvents can extract amyloids from FFPE specimens by converting their secondary structure. This method could eliminate the LMD step and simplified amyloid typing by MS analysis. •DMSO, DMF, methanol, TFE and HFIP can extract Aβ specifically from the FFPE brain of a Alzheimer' disease mouse model.•DMSO can extract SAA specifically from a FFPE section of AA amyloidosis.•Secondary structures of Aβ and transthyretin converted from β-sheet to α-helix in TFE.
| S-EPMC7046812 | biostudies-literature
Project description:Current aquatic chemical testing guidelines recognise that solvents can potentially interfere with the organism or environmental conditions of aquatic ecotoxicity tests and therefore recommend concentration limits for their use. These recommendations are based on evidence of adverse solvent effects in apical level tests. The growing importance of sub-apical and chronic endpoints in future test strategies, however, suggests the limits may need re-assessment. To address this concern, microarrays were used to determine the effects of organic solvents, dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), upon the transcriptome of zebrafish (Danio rerio) embryos. Embryos were exposed for 48 h to 0.025 and 0.1 ml/L DMF or DMSO. Effects on survival and development after 24 and 48 h were assessed microscopically with no effects on mortality or morphology. However, analysis of 48-h embryonic RNA revealed large numbers of differentially expressed genes for both solvent at both concentrations. The enrichment of differentially expressed genes was found for metabolic, development and other key biological processes, some of which could be linked to observed morphological effects at higher solvent concentrations. These findings emphasise the need to remove or lower as far as possible, the concentrations of solvent carriers in ecotoxicology tests.
2011-10-06 | GSE32593 | GEO
Project description:While glycoproteins are abundant in nature, and changes in glycosylation occur in cancer and other diseases, glycoprotein characterization remains a challenge due to the structural complexity of the biopolymers. This paper presents a general strategy, termed GlyDB, for glycan structure annotation of N-linked glycopeptides from tandem mass spectra in the LC-MS analysis of proteolytic digests of glycoproteins. The GlyDB approach takes advantage of low-energy collision-induced dissociation of N-linked glycopeptides that preferentially cleaves the glycosidic bonds while the peptide backbone remains intact. A theoretical glycan structure database derived from biosynthetic rules for N-linked glycans was constructed employing a novel representation of branched glycan structures consisting of multiple linear sequences. The commonly used peptide identification program, Sequest, could then be utilized to assign experimental tandem mass spectra to individual glycoforms. Analysis of synthetic glycopeptides and well-characterized glycoproteins demonstrate that the GlyDB approach can be a useful tool for annotation of glycan structures and for selection of a limited number of potential glycan structure candidates for targeted validation.
| S-EPMC2557434 | biostudies-literature
Project description:Solvent evaporation is often used in the sample preparation procedure for the determination of organic pollutants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organic pesticides. Because of the loss of analyte during this step, a high-boiling solvent, i.e., a keeper, is often added to the extract before evaporation. However, there are almost no basic studies found in the literature on the selection of keepers for the appropriate type of analytes (keepers are usually selected only on the basis of information provided by various recommendations). In this work, the effect of several keepers (isooctane, toluene, nonane, octanol, dodecane) on the recovery of various analytes (PAHs, PCBs, organic pesticides) was evaluated (during evaporation in a stream of nitrogen, at 40 °C). The analysis of the results obtained for the tested compounds shows that 1-octanol is a universal keeper for compounds with low volatility, i.e., PCBs (average recovery: 97.6%), organochlorine pesticides (average recovery: 95.0%), organophosphorus pesticides (OPPs; average recovery: 99.7%) and higher mass PAHs (average recovery: 91.9%). The use of isooctane as a keeper yields high recoveries for PAHs, regardless of their volatility (average recovery: 95.5%). When using 1-octanol or dodecane as a keeper, the reversed solvent effect (during GC analysis) was noted in relation to volatile analytes causing the distortion of their peaks. Additionally, the phenomenon of loss of some analytes (e.g., OPPs) was observed during evaporation without heating the vials. However, in the case of PCBs, organochlorine pesticides (OCPs) and o-hydroxybiphenyl, evaporation under such conditions yields recoveries greater than or equal to 90.0%. The results presented in this work can help in finding a suitable keeper for a specific group of analytes or an alternative to the commonly used one, especially in the case of recovery problems.
| S-EPMC7582485 | biostudies-literature
Project description:Mass spectrometric analysis of intact glycopeptides can reveal detailed information about glycosite, glycan structural features, and their heterogeneity. Sialyl glycopeptides can be positively, negatively, or neutrally charged depending on pH of their buffer solution and ionization conditions. To detect sialoglycopeptides, a negative-ion mode mass spectrometry may be applied with a minimal loss of sialic acids, although the positively charged or neutral glycopeptides may be excluded. Alternatively, the sialyl glycopeptides can be identified using positive-ion mode analysis by doping a high concentration of sodium salts to the analytes. Although manipulation of unmodified sialoglycopeptides can be useful for analysis of samples, less than optimal ionization, facile loss of sialyl and unfavorable ionization of accompanying non-sialyl peptides make such strategies suboptimal. Currently available chemical derivatization methods, while stabilizing for sialic acid, mask sialic acid linkage configuration. Here, we report the development of a novel approach to neutralize sialic acids via sequentially chemical modification that also reveals their linkage configuration, often an important determinant in biological function. This method utilizes several components to facilitate glycopeptide identification. These include the following: solid phase derivatization, enhanced ionization of sialoglycopeptides, differentiation of sialic acid linkage, and enrichment of the modified glycopeptides by hydrophilic interaction liquid chromatography. This technology can be used as a tool for quantitative analysis of protein sialylation in diseases with determination of sialic acid linkage configuration. Graphical Abstract ᅟ.
| S-EPMC6744383 | biostudies-literature
Project description:A new class of glycan-reactive HIV-neutralizing antibodies, including PG9 and PG16, has been recently discovered that seem to recognize previously uncharacterized glycopeptide epitopes on HIV-1 gp120. However, further characterization and reconstitution of the precise neutralizing epitopes are complicated by the heterogeneity of glycosylation. We report here the design, synthesis and antigenic evaluation of new cyclic V1V2 glycopeptides carrying defined N-linked glycans at the conserved glycosylation sites (Asn160 and Asn156 or Asn173) derived from gp120 of two HIV-1 isolates. Antibody binding studies confirmed the necessity of a Man?GlcNAc? glycan at Asn160 for recognition by PG9 and PG16 and further revealed a critical role of a sialylated N-glycan at the secondary site (Asn156 or Asn173) in the context of glycopeptides for antibody binding. In addition to defining the glycan specificities of PG9 and PG16, the identified synthetic glycopeptides provide a valuable template for HIV-1 vaccine design.
| S-EPMC3730851 | biostudies-literature
Project description:Aberrant glycosylation has been linked to many different cancer types. The blood-brain barrier (BBB) is a region of the brain that regulates the entrance of ions, diseases, toxins, and so on. However, in breast cancer metastasis, the BBB fails to prevent the crossing of the cancer cells into the brain. Here we present a study of identifying and quantifying the glycosylation of six breast and brain cancer cell lines using hydrophilic interaction liquid chromatography (HILIC) and electrostatic repulsion liquid chromatography (ERLIC) enrichments and LC-MS/MS analysis. Qualitative and quantitative analyses of N-linked glycosylation were performed by both enrichment techniques for individual and complementary comparison. Potential cancer glycopeptide biomarkers were identified and confirmed by chemometric and statistical evaluations. A total of 497 glycopeptides were characterized, of which 401 were common glycopeptides (80.6% overlap) identified from both enrichment techniques. HILIC enrichment yielded 320 statistically significant glycopeptides in 231BR relative to the other cell lines out of 494 unique glycopeptides, and sequential HILIC-ERLIC enrichment yielded 214 statistically significant glycopeptides in 231BR compared with the other cell lines out of 404 unique glycopeptides. The results provide the first comprehensive glycopeptide listing for these six cell lines.
| S-EPMC5748158 | biostudies-other
Project description:Current aquatic chemical testing guidelines recognise that solvents can potentially interfere with the organism or environmental conditions of aquatic ecotoxicity tests and therefore recommend concentration limits for their use. These recommendations are based on evidence of adverse solvent effects in apical level tests. The growing importance of sub-apical and chronic endpoints in future test strategies, however, suggests the limits may need re-assessment. To address this concern, microarrays were used to determine the effects of organic solvents, dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), upon the transcriptome of zebrafish (Danio rerio) embryos. Embryos were exposed for 48 h to 0.025 and 0.1 ml/L DMF or DMSO. Effects on survival and development after 24 and 48 h were assessed microscopically with no effects on mortality or morphology. However, analysis of 48-h embryonic RNA revealed large numbers of differentially expressed genes for both solvent at both concentrations. The enrichment of differentially expressed genes was found for metabolic, development and other key biological processes, some of which could be linked to observed morphological effects at higher solvent concentrations. These findings emphasise the need to remove or lower as far as possible, the concentrations of solvent carriers in ecotoxicology tests. Balanced loop design consisting of five separate conditions - two exposure concentrations for each of the two solvents and a control treatment - using four replicates for each condition all of which were dye swapped, resulting in a total of twenty independant samples on twenty arrays.
2011-10-05 | E-GEOD-32593 | biostudies-arrayexpress
Project description:As one of the most important and fruitful methods, supramolecular self-assembly has a significant advantage in designing and fabricating functional soft materials with various nanostructures. In this research, a low-molecular-weight gelator, N,N'-di(pyridin-4-yl)-pyridine-3,5-dicarboxamide (PDA-N4), was synthesized and used to construct self-assembled gels via a solvent-mediated strategy. It was found that PDA-N4 could form supramolecular gels in mixed solvents of water and DMSO (or DMF) at high water fraction (greater than or equal to 50%). By decreasing the water fraction from 50% to 30%, the gel, suspension and solution phases appeared successively, indicating that self-assembled aggregates could be efficiently modulated via water content in organic solvents. Moreover, the as-prepared PDA-N4 supramolecular gels not only displayed solid-like behavior, and pH- and thermo-reversible characteristics, but also showed a solution-gel-crystal transition with the extension of aging time. Further analyses suggested that both the crystal and gel had similar assembled structures. The intermolecular hydrogen bonding between amide groups and the π-π stacking interactions between pyridine groups played key roles in gel formation. Additionally, the release behavior of vitamin B12 (VB12) from PDA-N4 gel (H2O/DMSO, v/v = 90/10) was evaluated, and the drug controlled release process was consistent with a first-order release mechanism. The human umbilical venous endothelial cell culture results showed that the PDA-N4 xerogel has good cytocompatibility, which implied that the gels have potential biological application in tissue engineering and controlled drug release.
| S-EPMC8695936 | biostudies-literature