Isomeric separation of permethylated glycans by extra-long reversed-phase liquid chromatography (RPLC)-MS/MS

Abstract

Glycosylation is known as a critical biological process that can largely affect the properties and the functions of proteins. Glycan isomers have been shown to be involved in a variety of disease progressions. However, the separation and identification of glycan isomers has been a challenge for years due to the microheterogeneity of glycan isomeric structures. Therefore, effective and stable techniques have been investigated over the last few decades to improve isomeric separations of glycans. RPLC has been widely used in biomolecule analysis because of its extraordinary reproducibility and reliability in retention time and separation resolution. However, so far, no studies have achieved high resolution of glycan isomers using this technique. In this study, we focused on further boosting the isomeric separation of permethylated glycans using a 500 mm reversed-phase LC column. To achieve better resolutions on permethylated glycans, different LC conditions were optimized using glycan standards, including core- and branch-fucosylated N-glycan isomers and sialic acid linked isomers, which were both successfully separated. Then, the optimal separation strategy was applied to achieve separations of N- and O-glycan isomers derived from model glycoproteins, including bovine fetuin, ribonuclease B and κ-casein. Baseline separations were observed on multiple sialylated linkage isomers. However, the separation performance of high-mannose isomers needs further improvement. The reproducibility and stability of this long C18 column was also tested by doing run-to-run, day-to-day and month-to-month comparisons of retention times on multiple glycans and the %RSD was found less than 0.92%. Finally, we applied this approach to separate glycan isomers derived from complex biological samples, including blood serum and cell lines, where baseline separations were attained on several isomeric structures. Compared to the separation efficiency of PGC and MGC columns, the RPLC C18 column provides lower resolution but more robust reproducibility, which makes it a good complementary alternative for isomeric separations of glycans.

Figure 1.

Extracted Ion Chromatograms (EICs) of permethylated core- and branch-fucosylated glycan standards (HexNAc₄Hex₄Fuc₁) using (A) 150 mm C18 column under 55°C (B) 500 mm C18 column under 60°C. (The insets on either side of the peaks provide the MS/MS spectra for structural identification.) Symbols: , N-acetylglucosamine (GlcNAc); , Galactose (Gal); , Fucose (Fuc); , Mannose (Man); , Glucose (Glc); , N-acetylneuraminic acid (NeuAc/Sialic Acid).

Figure 2.

EICs of permethylated sialic acid linkage glycan standards with composition of (A, B) HexNAc₄Hex₅NeuAc₁ and (C, D) HexNAc₄Hex₅Fuc₁NeuAc₁ mixed at different ratios (α2,6: α2,3 = 3: 1 and α2,6: α2,3 = 1: 3) for linkage isomer identification. Symbols: as shown in Figure 1.

Figure 3.

(A) TIC of permethylated sialic acid linkage N-glycans derived from bovine fetuin, and EICs of (B) HexNAc₄Hex₅NeuAc₁ and (E) HexNAc₄Hex₅NeuAc₂ analyzed on the 500 mm C18 column. (C, F) EICs of permethylated HexNAc₄Hex₅NeuAc₁ and HexNAc₄Hex₅NeuAc₂ after the α2,3 neuraminidase digestion. (D, G) Distributions of isomers from NMR (blue) and LC/MS (orange). Symbols: as shown in Figure 1.

Figure 4.

Reproducibility and stability test of the 500 mm C18 column by performing month-to-month comparisons across three months. Demonstrated by N-glycans with compositions of (A) HexNAc₅Hex₆NeuAc₃ and (B) HexNAc₅Hex₆NeuAc₄ derived from bovine fetuin. Symbols: as shown in Figure 1.

Figure 5.

Baseline separations of permethylated isomeric O-glycan structures. (A) 3’-sialyllactose and 6’-sialyllactose, (B) HexNAc₂Hex₂NeuAc₂ derived from bovine fetuin and (C) HexNAc₁Hex₁NeuAc₁ derived from κ-casein. Symbols: as shown in Figure 1.

Figure 6.

Direct comparisons of EICs of permethylated N-glycans released from breast cancer cell line MDA-MB-231BR and brain cancer cell line CRL-1620. (A) HexNAc₄Hex₃Fuc₁, (B) HexNAc₅Hex₃Fuc₁, (C) HexNAc₄Hex₅NeuAc₁, and (D) HexNAc₄Hex₅NeuAc₂. The insets illustrate the relative abundance of isomers from the two different cell lines. **: p<0.01, ***: p<0.001.