What is N-and O-Glycosylation Site Occupation?
N-and O-glycosylation site occupation refers to the extent to which specific amino acid residues within a protein are modified by the addition of carbohydrate moieties through N-glycosylation or O-glycosylation. These modifications play crucial roles in protein folding, stability, localization, and function in various biological processes.
- N-glycosylation: This type of glycosylation occurs on asparagine (N) residues within the consensus sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. The addition of complex carbohydrate chains to these asparagine residues can significantly influence protein structure and function. The occupancy of N-glycosylation sites refers to the proportion of asparagine residues within this consensus sequence that are actually glycosylated.
- O-glycosylation: O-glycosylation, on the other hand, involves the attachment of carbohydrate moieties to the hydroxyl group of serine (S) or threonine (T) residues within a protein. Unlike N-glycosylation, O-glycosylation sites do not have a well-defined consensus sequence, making their prediction more challenging. Similar to N-glycosylation, the occupancy of O-glycosylation sites refers to the proportion of serine or threonine residues that are glycosylated.
Determining the site occupancy of N-and O-glycosylation is crucial for understanding the functional consequences of glycosylation on protein behavior. High occupancy at specific sites may suggest important functional roles for those modifications, such as stabilizing protein structure or influencing protein-protein interactions. Conversely, low occupancy may indicate that glycosylation at those sites is more variable or context-dependent.
Several experimental techniques can be employed to assess glycosylation site occupancy, including mass spectrometry-based approaches, glycan microarrays, and site-directed mutagenesis coupled with glycosylation site mapping. These methods allow researchers to identify glycosylation sites within a protein and quantify the extent to which these sites are occupied by carbohydrate moieties under different physiological or pathological conditions.
Creative Proteomics offers comprehensive N- and O-glycosylation site occupation analysis services, empowering researchers to unravel the complexities of glycoproteins with precision and efficiency.
N-and O-Glycosylation Site Occupation Analysis Services in Creative Proteomics
Glycosylation Site Identification – We utilize Mass Spectrometry (MS) to identify the exact sites of N- and O-linked glycosylation within your protein sample. This involves a combination of enzymatic and chemical methods to prepare glycopeptides for analysis and uses high resolution tandem MS to detect the modified peptides.
Glycosylation Site Occupancy – After identifying the sites of glycosylation, we quantify the proportion of each site that is occupied by a glycan. This is achieved via targeted MS data acquisition and analysis.
Glycan Structure Analysis – We perform a comprehensive analysis of the glycan structures attached to each site. This is achieved through high-resolution MS and tandem MS analysis combined with exoglycosidase digestions.
Site-Specific Glycan Heterogeneity Profiling – Utilizing our sophisticated analytical techniques, we can identify and quantify the different glycan structures at individual glycosylation sites, providing a comprehensive profile of site-specific glycan heterogeneity.
Structural Characterization of the Glycosylation Site – We employ the use of various Spectral database and bioinformatic tools to interpret the MS data, leading to the structural characterization of each glycosylation site.
Impact Assessment – While analyzing the glycosylation site occupancy and heterogeneity, we also evaluate the potential impact of the glycosylation on the function and stability of the protein, which can provide significant insights into the protein's biological function.
Detailed Report – In the end, you will receive a detailed report outlining the steps we took in the analysis, the results from each step, and our interpretations.
Technology Platform for N-and O-Glycosylation Site Occupation Analysis
Experimental Techniques:
a. Mass Spectrometry (MS): MS is a key technique for analyzing glycosylation sites. It involves ionizing analytes and measuring the mass-to-charge ratio of ions. For glycoproteins, MS can identify the masses of glycopeptides, providing information about the attached glycans and their occupancy.
b. Glycopeptide Enrichment: To enhance the detection of glycopeptides, enrichment techniques such as lectin affinity chromatography or hydrazide chemistry can be employed. These methods selectively capture glycopeptides from complex protein mixtures, increasing the sensitivity of glycosylation analysis.
c. Enzymatic Digestion: Proteins are typically enzymatically digested into peptides prior to MS analysis. Enzymes such as trypsin can cleave proteins at specific sites, generating peptides with glycosylation sites intact. This allows for the identification of glycopeptides by MS.
Data Analysis and Computational Tools:
a. Database Search: MS data is analyzed using database search algorithms to identify glycopeptides. These algorithms compare experimental MS spectra with theoretical spectra generated from databases of known protein sequences and glycan structures.
b. Glycan Identification: Software tools specifically designed for glycan identification can match experimental MS/MS spectra with glycan databases to determine the composition and structure of attached glycans.
c. Site Occupancy Calculation: Computational methods are used to calculate the occupancy of glycosylation sites based on the relative abundance of glycosylated and non-glycosylated peptides. These methods take into account factors such as peak intensity and spectral counts to estimate the proportion of occupied sites.
d. Statistical Analysis: Statistical techniques are applied to assess the reliability of glycosylation site occupancy measurements and identify significant differences between samples. This includes methods such as t-tests, ANOVA, or non-parametric tests depending on the nature of the data.
Integration and Visualization:
a. Data Integration: Results from MS analysis and computational tools are integrated to generate a comprehensive profile of glycosylation site occupancy for the protein of interest.
b. Visualization Tools: Data visualization tools such as heatmaps, bar charts, and interactive plots can be used to present the glycosylation site occupancy data in a clear and informative manner, facilitating interpretation and comparison between samples.
Sample Requirements for N-and O-Glycosylation Site Occupation Analysis
| Sample Type | Sample Volume/Quantity | Additional Information |
|---|---|---|
| Protein Extracts | 50-200 µg | Extracted from cell lysates, tissue homogenates, or biological fluids. |
| Whole Cell Lysates | 10-100 µg protein | Protein extracted from cultured cells or tissues. |
| Secreted Proteins | 100-500 µL | Proteins secreted into culture supernatants or bodily fluids. |
| Glycoprotein Isolates | 50-200 µg | Purified glycoproteins from biological samples or recombinant sources. |
| Membrane Proteins | 50-200 µg protein | Enriched membrane protein fractions from cells or tissues. |
| Serum/Plasma | 50-200 µL | Requires proper storage and handling to preserve glycoproteins. |
| Tissue Biopsies | 10-50 mg | Homogenized in appropriate lysis buffer for protein extraction. |
| Cell Pellets | 1-10 million cells | Requires cell lysis and protein extraction prior to analysis. |
| Urine | 100-500 µL | Requires careful handling and storage to preserve glycoproteins. |
| Cerebrospinal Fluid | 100-500 µL | Suitable for analysis of glycoproteins relevant to neurological disorders. |
| Saliva | 100-500 µL | May contain a diverse range of glycoproteins. |
| Milk | 100-500 µL | Analysis of milk glycoproteins is relevant to lactation and nutrition research. |
| Synovial Fluid | 100-500 µL | Analysis of glycoproteins in synovial fluid may be relevant to rheumatology. |
| Tears | 100-500 µL | Analysis of tear glycoproteins for ocular health research. |
| Feces | 50-200 mg | Suitable for analysis of fecal glycoproteins in microbiome research. |
Differences Between Services in N- and O-Linked Glycosylation Mapping
| Service Name | Difference |
|---|---|
| N- and O-Glycan Profiling Service | - This service is used to analyze the overall composition and structural characteristics of N- and O-glycans in a sample. - Techniques such as mass spectrometry and chromatography are employed to determine the types, structures, and relative abundances of different N- and O-glycans in the sample. |
| N-and O-Glycosylation Site Services | - This service aims to determine the N- and O-glycosylation sites within proteins, i.e., identifying which amino acid residues are glycosylated. - Techniques such as mass spectrometry, sequence analysis, etc., are utilized to pinpoint the specific locations of glycosylation within proteins. |
| N- and O-Glycan Linkage Analysis | - This service is employed to study the linkage patterns and structures between N- and O-glycans. - Techniques such as mass spectrometry and liquid chromatography are used to determine the linkage positions and types (e.g., β-1,4 linkage or α-1,6 linkage) between glycans. |
| N-and O-Glycosylation Site Occupation | - This service focuses on investigating the relative occupancy of N- and O-glycans within protein structures. - Techniques such as quantitative mass spectrometry and liquid chromatography are employed to determine the extent of occupancy of different glycan types under specific conditions, as well as the relative occupancy ratios between different glycans. |
Your sample's confidentiality will always be our top priority. Your results will be ready within the agreed timeframe, and if you have any questions, our team will be more than ready to assist you. Don't hesitate to reach out to us to find out more about our services.