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What is Mannose?
Mannose, a hexose sugar with the molecular formula C₆H₁₂O₆, is a critical building block in the biology of cells, particularly in glycosylation—a process where sugars are attached to proteins or lipids to form glycoproteins or glycolipids. This monosaccharide, while less abundant than glucose, plays a vital role in cellular functions, especially in protein folding, stability, and intracellular signaling. It is also an integral part of the structural and functional integrity of glycoproteins.
Mannose's involvement in N-linked glycosylation is especially important. In this pathway, mannose is incorporated into glycan chains attached to proteins, impacting the biological activity and half-life of glycoproteins. This makes mannose quantification and analysis essential for industries focused on biotechnology, pharmaceuticals, and biomaterials.
Why Need Quantitative Mannose Analysis?
- Biopharmaceutical Development: Many biopharmaceuticals, especially glycoprotein-based drugs like monoclonal antibodies, require stringent control over their glycan profiles, including mannose residues. The glycosylation pattern can affect a drug's efficacy, stability, and safety.
- Protein Quality Control: In protein engineering, ensuring the proper glycosylation of recombinant proteins (including correct mannose attachment) is critical for achieving the desired biological function.
- Functional Studies: Mannose plays a role in various biological pathways. Studying its presence and quantity in different glycoproteins helps in understanding its effect on protein-protein interactions, cell adhesion, and immune response.
Understanding the mannose content and structure within glycan chains provides crucial insights for optimizing biopharmaceutical products, improving quality control processes, and advancing scientific research.
Mannose Analysis Services Offered by Creative Proteomics
Mannose Quantification in Glycoproteins: We offer precise and sensitive quantification of mannose residues in glycoproteins. This service is ideal for biopharmaceutical companies needing to monitor the glycosylation patterns of their products.
N-Linked Glycan Profiling: We perform detailed analysis of N-linked glycans, identifying and quantifying mannose as part of the glycan structure. This service is invaluable for understanding the role of mannose in glycoprotein functionality and stability.
Monosaccharide Composition Analysis: Our monosaccharide composition analysis service provides a detailed breakdown of the sugar components in glycoproteins, including mannose. This helps researchers understand the complete glycan composition of a sample.
Enzymatic Assays for Mannose-Containing Glycans: Utilizing specific enzymes, we offer assays to analyze mannose-rich glycans, providing a focused approach to understanding mannose incorporation in biological molecules.
Mannose Metabolism Studies: This service enables the study of mannose metabolism in different biological samples, helping researchers to elucidate how mannose is processed in various cellular contexts and how it affects overall metabolic pathways.
Brochures
Metabolomics Services
We provide unbiased non-targeted metabolomics and precise targeted metabolomics services to unravel the secrets of biological processes.
Our untargeted approach identifies and screens for differential metabolites, which are confirmed by standard methods. Follow-up targeted metabolomics studies validate important findings and support biomarker development.
Download our brochure to learn more about our solutions.
Brochures
Glycomics Services
Glycomics, the study of glycans and their roles in biological systems, is critical for understanding processes like glycosylation, immune recognition, and cell signaling. At Creative Proteomics, we offer advanced platforms such as high-resolution mass spectrometry, glycan microarrays, and glycoproteomics to provide precise glycan profiling and analysis.
For detailed insights into our glycomics solutions and methodologies, download our Glycomics Service Brochure and discover how we can support your research.
Technology Platforms for Mannose Analysis
High-Performance Liquid Chromatography (HPLC)
HPLC is our primary tool for separating and quantifying mannose in glycoproteins and complex biological samples. We utilize Agilent 1260 Infinity II HPLC systems, known for their high sensitivity and resolution. This system provides rapid, accurate mannose quantification, essential for biopharmaceutical quality control.
Liquid Chromatography-Mass Spectrometry (LC-MS)
For detailed structural analysis, we use Thermo Scientific Q Exactive Orbitrap LC-MS, which combines high-resolution mass spectrometry with HPLC. This platform allows precise identification and structural characterization of mannose-containing glycans.
Capillary Electrophoresis (CE)
We leverage Beckman Coulter PA 800 Plus Capillary Electrophoresis for fast and efficient separation of monosaccharides, including mannose. CE is especially useful for analyzing small sample volumes while maintaining high resolution and speed.
Gas Chromatography-Mass Spectrometry (GC-MS)
For comprehensive monosaccharide profiling, our Agilent 7890B GC coupled with 5977B MS is used. After derivatization, GC-MS offers highly sensitive detection of mannose in various sample types, including complex polysaccharides and glycoproteins.
Sample Requirements for Mannose Analysis
| Sample Type | Required Amount | Preservation Conditions | Additional Notes |
|---|---|---|---|
| Purified Proteins/Glycoproteins | ≥ 50 µg | Store at -20°C | Avoid freeze-thaw cycles to maintain integrity |
| Serum or Plasma | ≥ 100 µL | Store at -80°C | Use cryovials and ship on dry ice |
| Cell Lysates | ≥ 100 µL | Store at -80°C | Provide details on buffer composition |
| Tissue Samples (Frozen) | ≥ 100 mg | Store at -80°C | Flash freeze immediately upon collection |
| Polysaccharides | ≥ 200 µg | Store at -20°C | Ensure samples are freshly prepared or lyophilized |
| Biological Fluids (e.g., urine) | ≥ 500 µL | Store at -80°C | Seal containers properly to avoid contamination |
| Recombinant Proteins | ≥ 50 µg | Store at -20°C | Provide information on protein concentration |
| Cell Culture Supernatants | ≥ 1 mL | Store at -80°C | Collect in sterile tubes and clarify by centrifugation |
Note: For best results, ensure that samples are freshly prepared or properly preserved to prevent degradation. Consult with our team for guidance on any unique sample types or specific project requirements.
PCA chart
PLS-DA point cloud diagram
Plot of multiplicative change volcanoes
Metabolite variation box plot
Pearson correlation heat map
Insect derived extra oral GH32 plays a role in susceptibility of wheat to Hessian fly
Journal: Scientific Reports
Published: 2021
Background
The Hessian fly (Mayetiola destructor) is an obligate parasite of wheat that causes significant economic damage through its interactions with the host plant. The larvae of this pest cannot feed by chewing tissue or sucking phloem sap, which necessitates the establishment of a nutrient-rich environment for their survival. The study investigates the role of the glycoside hydrolase MdesGH32, produced by the virulent larvae, which localizes within the leaf tissue. This enzyme possesses strong inulinase and invertase activity, facilitating the breakdown of inulin in the plant cell wall and converting sucrose into glucose and fructose. This process leads to the formation of nutrient-rich tissues that support larval development. The findings elucidate the molecular mechanisms by which Hessian fly larvae create a nutrient sink, promoting their susceptibility in wheat and potentially offering insights for pest management strategies.
Materials & Methods
1) Insect and Plant Materials
Mayetiola destructor (Hessian fly) Biotype L was sourced from USDA-ARS. Wheat lines included resistant ('Iris', 'Hamlet') and susceptible ('Newton', 'Centurk') varieties, along with nonhost Brachypodium distachyon (Bd21).
2) Plant Growth and Infestation
Wheat plants were grown in pots and infested with Hessian flies at the 1-leaf stage. Brachypodium seeds were grown in a 50:50 growing medium.
3) Tissue Collection
Larval samples were collected from wheat lines and Brachypodium at specified days after hatching (DAH). Crown tissues from infested and uninfested plants were harvested for analysis.
4) RNA Isolation and qRT-PCR
RNA was extracted using TRIzol and cDNA synthesized for qRT-PCR. Specific primers quantified MdesGH32, MdesGLUT, and cell wall-associated gene expressions.
5) Cloning and Gene Analysis
The MdesGH32 transcript was cloned, and genomic DNA was extracted for PCR amplification.
6) Protein Purification
Recombinant MdesGH32 was expressed in E. coli and purified using Ni2+ NTA resin. Total protein was extracted from Hessian fly larvae and plant tissues.
7) Immunodetection
Polyclonal antibodies against MdesGH32 were produced for immunoblotting and immunohistochemical analysis of protein localization in plant tissues.
8) Monosaccharide Quantification
Glucose and fructose levels in wheat seedlings were measured using UPLC-MS after tissue extraction.
9) Enzyme Activity Assays
Invertase, inulinase, and levanase activities were evaluated in larval proteins and recombinant MdesGH32 using substrate assays.
10) Neutral Red Staining
Neutral red staining assessed plant tissue integrity post-infestation, with staining intensity scored for analysis.
Results
MdesGH32 Structure and Function:
MdesGH32 has a predicted length of 1575 bp, encoding a 524 amino acid polypeptide with a molecular mass of 60.9 kDa. The protein features conserved domains typical of glycoside hydrolase family GH32, including a five-bladed β-propeller catalytic domain, indicating its enzymatic role.
Phylogenetic Analysis:
MdesGH32 clusters primarily with bacterial GH32 sequences, suggesting its origin from horizontal gene transfer. It shows a distant relationship with GH32 sequences from other insects, fungi, and plants.
Expression Levels:
Transcript levels of MdesGH32 significantly increase in virulent larvae feeding on susceptible wheat, peaking at 421-fold at 3 days after hatching (DAH). In contrast, avirulent larvae exhibit much lower expression levels.
Localization in Plant Tissue:
Immunohistochemical studies confirm the presence of MdesGH32 within the mesophyll cells and epidermis of wheat plants infested by larvae, indicating its extra-oral secretion.
Cell Wall Permeability:
Increased cell wall permeability is observed in susceptible wheat lines, suggesting that MdesGH32 contributes to the breakdown of cell wall polymers, facilitating nutrient absorption for the larvae.
Glucose and Fructose Levels:
Higher glucose levels are detected in susceptible wheat post-infestation, while fructose levels remain stable. The expression of TaFRK (fructokinase) in susceptible wheat correlates with the observed increases in fructose.
Wheat monosaccharide levels. (a) Glucose levels. (b) Fructose levels.
MdesGLUT Expression:
Transcripts of MdesGLUT, a glucose transporter, are elevated in virulent larvae, indicating the active transport of glucose across the larval midgut, essential for their growth.
Impact on Cell Wall-Related Genes:
Down-regulation of genes involved in cell wall biosynthesis is significant in susceptible wheat lines after infestation, demonstrating a negative impact on the plant's defense mechanisms.
How sensitive is mannose detection in your analysis services?
At Creative Proteomics, we use highly sensitive instruments like HPLC, LC-MS, and GC-MS, which can detect mannose in very low concentrations, often in the picomolar to nanomolar range. Our platforms are calibrated to detect both free mannose and mannose residues in glycoproteins, ensuring precise quantification even in complex samples. Sensitivity can depend on the sample type and preparation, so we recommend consulting with our team to tailor the analysis based on your specific needs.
What factors can affect the accuracy of mannose quantification in glycoproteins?
Several factors can influence the accuracy of mannose quantification, including sample purity, glycoprotein stability, and the presence of interfering substances like salts, detergents, or other carbohydrates. Proper sample preparation is crucial—impurities or degradation can skew results. We advise using highly purified samples and avoiding multiple freeze-thaw cycles, which can compromise glycan structure and affect mannose measurements. Additionally, our team employs rigorous controls and calibration standards to ensure high accuracy in every analysis.
What are the best practices for preparing glycoprotein samples for mannose analysis?
For optimal results in mannose analysis, glycoproteins should be freshly prepared and purified. We recommend using at least 50 µg of sample, stored at -20°C to preserve integrity, and avoiding repeated freeze-thaw cycles. Before submission, ensure that your samples are free from contaminants like salts or excessive buffer components, as these may interfere with the analysis process. If you're unsure about your sample quality or preparation protocol, our technical experts can provide guidelines tailored to your specific project.
Can mannose residues be selectively targeted in complex glycan structures?
Yes, using specialized enzymes such as endoglycosidases or exoglycosidases, we can selectively cleave and analyze mannose residues within complex glycan structures. This enzymatic approach allows us to isolate mannose-containing glycans and provide focused analysis of mannose incorporation. Combined with techniques like LC-MS or HPLC, this strategy ensures that we accurately quantify mannose while maintaining the integrity of the overall glycan profile.
How do you ensure reproducibility in mannose analysis across different samples?
Reproducibility is ensured through stringent quality control protocols, including the use of internal standards, consistent calibration of instruments, and batch-specific controls. All our assays are validated, and we conduct multiple runs where necessary to verify consistency in mannose detection and quantification. By normalizing results across runs and carefully managing sample handling, we ensure high reproducibility even when analyzing large sample sets or different biological matrices.
Can you perform mannose analysis on low-abundance glycoproteins?
Yes, we have the capability to analyze low-abundance glycoproteins using highly sensitive techniques like LC-MS with Orbitrap technology. By optimizing sample preparation and using enrichment methods for glycoproteins, we can detect mannose residues in samples where the target glycoproteins are present in low quantities. Our high-resolution instruments are designed to work with minimal sample volumes, ensuring you can get accurate results even with limited material.
How do you handle mannose-containing polysaccharides or more complex glycans?
Mannose-containing polysaccharides or highly branched glycans require a tailored approach. We utilize a combination of chemical and enzymatic digestion methods to break down these complex structures into their monosaccharide components for precise analysis. Techniques such as GC-MS and HPLC are particularly effective in identifying and quantifying mannose within these complex structures. This approach allows us to provide a detailed breakdown of glycan composition, even in structurally diverse samples.
What are the potential sources of error in mannose analysis, and how do you minimize them?
Potential sources of error in mannose analysis include sample degradation, contamination, and instrument variability. To minimize these risks, we implement strict sample handling protocols, including temperature-controlled storage and shipment, to preserve sample integrity. In addition, our instruments are calibrated regularly, and we use high-purity reagents to prevent contamination. Data are reviewed by experts to identify any anomalies, and replicate analyses are performed when necessary to confirm accuracy.
Summative and ultimate analysis of live leaves from southern US forest plants for use in fire modeling.
Matt, Frederick J., Mark A. Dietenberger, and David R. Weise.
Journal: Energy & Fuels
Insect derived extra oral GH32 plays a role in susceptibility of wheat to Hessian fly.
Subramanyam, Subhashree, et al.
Journal: Scientific Reports
Year: 2021
https://doi.org/10.1038/s41598-021-81481-4
