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High-throughput Profiling of Permethylated Bovine Fetuin N-glycans

Analysis of Permethylated Sialylated N-glycans

September 30, 2021

Bovine fetuin is often used as a model glycoprotein for glycosylation studies in bio-therapeutics. Previous studies reveal that the glycan elution profile in bovine fetuin consists of a series of neutral glycans first, followed by monosialylated, disialylated, trisialylated, tetrasialylated, and finally pentasialylated species. Sialylated N-glycans play essential roles in the immune system, pathogen recognition, and cancer. However, the sialic acids are labile, and hence are difficult to analyze. Moreover, the difference in the ionization efficiency of the sialylated glycans and neutral glycans makes a high throughput glycan profiling attempt more challenging.

Permethylation is a glycan derivatization technique where methyl groups (CH3) are added to all hydroxyl and N-acetyl groups of a glycan structure. This derivatization technique is popular in analyzing sialylated glycans because it stabilizes the sialic acids and thus enhances the ionization efficiency.

In a recent analysis, bovine fetuin N-glycans were extracted using Peptide -N-Glycosidase F (PNGaseF), an amidase that cleaves between the innermost GlcNAc and asparagine residues of N-linked glycoproteins. The released N-glycans were permethylated before injecting into mass spectrometry. High-Resolution mass spectrometry was performed in positive ion mode using Thermo Scientific Orbitrap Fusion Tribrid Mass Spectrometer.

The raw data from the LC-MS experiment was analyzed using SimGlycan® software. Using 1% MS1 peak threshold intensity, an absolute intensity value of 10000 as the minimum peak height, 0.1 min – 2.0 min as acceptable peak width, availability of a minimum of three isotopic peaks to validate an LC compound, SimGlycan created a peak list including all possible compounds. The software clustered the MS/MS scans for the detected compounds. MS/MS database search was performed using 5 ppm and 10 ppm as precursor ion and product ion tolerances, and sodiated species as predominant ion species. The database search annotated 43 LC compounds with 10 unique composition structures.

Schematic representation of high throughput profiling of permethylated N-glycans from Bovine fetuin using SimGlycan Software

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