Can LC-MS Detect Enzymatic Glycosylation Modifications
Liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for detecting enzymatic glycosylation modifications. Glycosylation, a prevalent post-translational modification (PTM) of proteins, is crucial in various biological processes such as cellular signaling, protein folding, and immune recognition. LC-MS is especially advantageous in glycosylation analysis because it provides comprehensive data on glycosylation sites, including the glycosylation type and glycan chain structure.
LC-MS Procedure for Glycosylation Detection
1. Protein Digestion
Proteins are initially digested into smaller peptides using specific enzymes, such as trypsin.
2. Enrichment of Glycosylated Peptides
Due to the low abundance of glycosylated peptides in protein samples, enrichment techniques like lectin affinity chromatography are employed to enhance detection sensitivity and specificity.
3. Liquid Chromatography Separation
Glycosylated peptides are separated based on their hydrophobicity, charge, and other properties using a liquid chromatography system.
4. Mass Spectrometry Analysis
The separated peptides are then analyzed by mass spectrometry, which provides precise mass measurements, identifies glycosylation via mass shifts, and further examines glycan chain structures and compositions.
5. Data Analysis
Specialized software and databases are utilized to analyze data, identifying glycosylation sites and glycan chain types.
Advantages of LC-MS in Glycosylation Analysis
1. High Sensitivity and Specificity
It enables the detection of trace glycosylated peptides with exceptional specificity.
2. Quantitative Analysis
Beyond qualitative identification, LC-MS allows for relative or absolute quantification to evaluate glycosylation changes under varying conditions.
3. Structural Characterization
It yields detailed structural insights into glycan chains, including sugar types and linkage modes.
MtoZ Biolabs offers comprehensive LC-MS/MS services for glycosylation site and glycan type analysis. Our approach involves using multiple enzymes to extensively digest the target protein, ensuring complete sequence coverage. Post glycan cleavage, we analyze the glycosylation sites within the protein. Upon identifying glycosylated peptides, the protein is redigested, retaining the glycan on the peptide. During LC-MS/MS analysis, we employ combined ETD, HCD, and CID fragmentation to procure extensive secondary fragmentation data. Software-assisted analysis alongside manual verification of glycosylated peptide spectra provides precise information on glycosylation sites and glycan structures.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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