Principle of N-Glycan Profiling Based on MALDI-TOF-MS

Glycosylation refers to the process of carbohydrates binding to proteins, lipids, or other biomolecules, playing a crucial role in cellular signaling, immune responses, and protein stability. N-glycosylation, a major form of glycosylation, involves oligosaccharide chains attached to amino acid residues. In recent years, N-glycan analysis based on Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) has become a focus of attention for researchers.

 

The fundamental principle of MALDI-TOF-MS is to convert samples into gas-phase ions through matrix-assisted laser desorption and ionization, followed by mass analysis using time-of-flight mass spectrometry. The workflow primarily includes the following steps:

 

1. Sample Preparation

First, extract protein samples containing N-glycans and enzymatically cleave (e.g., using PNGase F) the glycan chains from glycosylated sites. This process typically involves different enzymes and chemical reagents to ensure comprehensiveness and accuracy.

 

2. Matrix Selection

Choose an appropriate matrix (e.g., α-cyano-4-hydroxycinnamic acid, CHCA) and mix it with the sample. The role of the matrix is to absorb laser energy and assist in the rapid evaporation and ionization of sample molecules upon laser irradiation.

 

3. Laser Irradiation

The mixture is spotted onto a MALDI target plate and dried; then, laser irradiation is used on the sample. The laser energy allows matrix molecules to absorb energy and convert into gas-phase ions while desorbing and ionizing the sample molecules.

 

4. Ionization and Flight

The desorbed ions are accelerated in an electric field and enter the time-of-flight mass spectrometry analyzer. Depending on the mass-to-charge ratio (m/z) of the ions, ions of different masses will produce varying flight times in the mass spectrometer.

 

5. Data Analysis

The detector of the time-of-flight mass spectrometer records the arriving ions and generates a mass spectrum. By analyzing the ions' mass in the spectrum, the composition and structure of the N-glycans can be inferred.

 

MALDI-TOF-MS possesses multiple advantages in N-glycan analysis, including high sensitivity, rapid analysis, and good resolution. However, it also has certain limitations, such as weaker detection capability for low-abundance components in complex samples and potential impacts on result accuracy due to sample selectivity and molecular ionization efficiency.