MS-based glycomics and glycoproteomics methods enabling isomeric characterization

Abstract

Glycosylation is one of the most significant and abundant posttranslational modifications in mammalian cells. It mediates a wide range of biofunctions, including cell adhesion, cell communication, immune cell trafficking, and protein stability. Also, aberrant glycosylation has been associated with various diseases such as diabetes, Alzheimer's disease, inflammation, immune deficiencies, congenital disorders, and cancers. The alterations in the distributions of glycan and glycopeptide isomers are involved in the development and progression of several human diseases. However, the microheterogeneity of glycosylation brings a great challenge to glycomic and glycoproteomic analysis, including the characterization of isomers. Over several decades, different methods and approaches have been developed to facilitate the characterization of glycan and glycopeptide isomers. Mass spectrometry (MS) has been a powerful tool utilized for glycomic and glycoproteomic isomeric analysis due to its high sensitivity and rich structural information using different fragmentation techniques. However, a comprehensive characterization of glycan and glycopeptide isomers remains a challenge when utilizing MS alone. Therefore, various separation methods, including liquid chromatography, capillary electrophoresis, and ion mobility, were developed to resolve glycan and glycopeptide isomers before MS. These separation techniques were coupled to MS for a better identification and quantitation of glycan and glycopeptide isomers. Additionally, bioinformatic tools are essential for the automated processing of glycan and glycopeptide isomeric data to facilitate isomeric studies in biological cohorts. Here in this review, we discuss commonly employed MS-based techniques, separation hyphenated MS methods, and software, facilitating the separation, identification, and quantitation of glycan and glycopeptide isomers.

Keywords: MS-based isomeric characterization; glycan isomers; glycopeptide isomers.

Figure 1.

Schematic of two-step derivatization of sialic acid linkage isomers. A α2,6-linked sialic acid forms a stable dimethylamide through EDC, HOBt, and dimethylamine reactions in the first step, and keeps the same structure in the second step. B α2,3-linked sialic acid forms an unstable lactone in the first step, and then convert to a stable amide in the second step. C Mass spectra of in situ derivatized (top) and native (bottom) N-glycans derived from leiomyosarcoma FFPE tissue showing the induced mass shift of +28.031 Da between α2,3- and α2,6-linked sialic acids after derivatization, while native N-glycans are detected with additional neutral proton-sodium exchange. Symbols: green circle represents mannose, yellow circle represents galactose (Gal), blue square represents N-acetylglucosamine (GlcNAc), yellow square represents N-acetylgalactosamine (GalNAc), red triangle represents fucose, purple diamond represents N-acetylneuraminic acid (NeuAc), Reproduced with the permission from ref (Holst et al. 2016).

Figure 2.

Sialic acid linkage and expression differences between tissue types in an advanced-stage pancreatic ductal adenocarcinoma (PDAC) tumor tissue. High-resolution (40 μm) MALDI-IMS of AA-stabilized sialylated N-glycans. A, annotated H&E staining of late-stage PDAC tumor. B, the overlay image of three N-glycan structures (depicted individually in panels C–E) localized to adjacent normal tissue (purple, Hex9HexNAc2 m/z 1905.6339), tumor stroma (green, Hex5dHex1HexNAc4NeuAc1(2,3) m/z 2099.7507), and adenocarcinoma (red, Hex₅dHex₂HexNAc₅NeuAc₁(2,6) m/z 2476.9193). C, high-mannose glycan structures tended to associate with normal adjacent tissue. N-glycan structures in normal tissue lacked significant sialic acid expression as compared with tumor tissue. D, α2,3-oriented sialic acid–decorated N-glycan structures localized to tumor stroma regions. E, adenocarcinoma-associated N-glycans were predominantly α2,6-sialylated although α2,3 vs. α2,6 sialylation of the same base structures drove different intratumor localization. These same α2,6-sialylated structures were also present within pancreatic intraepithelial neoplasia (Pan-IN) lesions. Symbols as in Figure 1. Reprinted with the permission from ref (McDowell et al. 2020).

Figure 3.

(A) MSⁿ defines sialic acid linkage position for the HexNAc₄Hex₅Neu5Ac₂ N-glycan derived from HIV gp120. (Bi) CID spectra (30% collision energy) of protonated 1 “purple” and 2 “green”, the precursor is labeled P. (Bii) IRMPD spectra of protonated 1“purple” and 2 “green”. Photofragmentation yield (dots), and 5 points FT rolling averaging (line). Symbols as in Figure 1. (A) Reproduced with the permission from ref (Kurz et al. 2021) and (B) reproduced with the permission from ref (Depraz Depland et al. 2018).

Figure 4.

Isomeric separation of glycans derived from different samples following the 96-well plate sample preparation method using nanoPGC-LC-MS/MS. (A) Combined EICs of N-glycans derived from bovine fetuin. Sialic acid linkage isomers were efficiently resolved on PGC column. (B) Combined EICs of N-glycans derived from NMuMG cells. Isomeric separation of sialylated N-glycan isomers was achieved on biological samples. (C) Combined EICs of O-glycans derived from NMuMG cells. Isomeric separation of two pairs of O-glycan isomers were observed, as indicated by purple and orange peaks. Symbols as in Figure 1. Reproduced with the permission from ref (Zhang et al. 2020).

Figure 5.

Biantennary standard glycan structures in gg conformation optimized using the PBE-D3/SV method in the COSMO/acetonitrile environment. Representations (A) and (C) show the polarization charges and CS-NPSA values, and (B) and (D) the polar atoms in red, nonpolar atoms contributing to the NPSA in blue, and buried nonpolar atoms that do not contribute to the NPSA in yellow. The core-fucosylated structure is shown in (A) and (B), while the branch-fucolyslated structure is shown in (C) and (D). Linear regression correlating experimentally determined retention times with CS-NPSA values for the core- and branch-fucosylated isomers and the 2,3- and 2,6-sialylated isomers in gg conformation using the PBE-D3/SV approach is shown in (E). Symbols as in Figure 1. Reproduced with the permission from ref (Dhakal et al. 2021).

Figure 6.

(A) EIC of the glycan F1A2G1 with galactose attached to different branches, which were released from human blood serum. (B) CID MS/MS spectrum of the isomeric structure eluted at 32.8 min, with molecular modeling of the structure. (C) CID MS/MS spectrum of the isomeric structure eluted at 34.1 min, with molecular modeling of the structure. (D) EIC of permethylated bi-antennary bi-sialylated glycans released from bovine fetuin. (E) EIC of F1A2G1 with isomers resulting from branch and core fucosylation. Symbols as in Figure 1. Reproduced with the permission from ref (Zhou et al. 2017).

Figure 7.

Separation of isomeric permethylated glycans derived from MDA-MB231, MDA-MB-231BR, and CRL-1620 on the MGC column (A) Separation of HexNAc₃Hex₃DeoxyHex₁. (B) Separation of HexNAc₄Hex₅NeuAc₂. (C) Separation of HexNAc₂Hex₈. (D) Separation of HexNAc₄Hex₃DeoxyHex₁. (E) Separation of HexNAc₄Hex₅NeuAc₁. (F) Separation of HexNAc₄Hex₅DeoxyHex₁NeuAc₂. Symbols as in Figure 1. Reproduced with permission from ref (Gautam et al. 2021).

Figure 8.

Extracted ion chromatograms of (A) O-glycopeptide with EAISPPDAASAAPLR backbone from EPO, and (B, C, D) tri-antennary N-glycopeptides containing LVPVPITNATLDR backbone from AGP, separated using a C18 at 30°C, 40°C, 50°C, and 60 °C. Symbols as in Figure 1. Reproduced with the permission from ref (Ji et al. 2019).

Figure 9.

Normalized EIC chromatograms of A2G2F1 glycoforms of SWPAVGN¹⁸⁷CSSALR (A–C) and ALPQPQN⁴⁵³VTSLLGCTH (D–F) in different HILIC columns. PEP1 refers to SWPAVGN¹⁸⁷CSSALR, and PEP2 to ALPQPQN⁴⁵³VTSLLGCTH. Symbols as in Figure 1. Reprinted with the permission from ref (Molnarova and Kozlík 2020).

Figure 10.

Isomeric separation of glycopeptides composed of different peptide backbones, antennary types, and degrees of sialylation from AGP on PGC. Glycopeptide structures were from glycosylation sites ⁵⁶Asn (A) and ¹⁰³Asn (B). Symbols as in Figure 1. Reprinted with the permission from ref (Zhu et al. 2020).

Figure 11.

(A) Arrival time distributions (ATDs) for Lewis and blood group oligosaccharides measured as sodium adducts. (B) SLIM SUPER IM separation of human milk oligosaccharide isomers LNT and LNnT (31.5 M “top” and 45 m “down”). (C) Differentiation of sialic acid linkage for the N-glycopeptide EVFVHPN-[H5N4S1(α2,3 or α2,6)]YSK using CID fragmentation and subsequent IM-MS analysis. Symbols as in Figure 1. (A) Reproduced with the permission from ref (Hofmann et al. 2017), (B) reproduced with the permission from (Nagy et al. 2018), and (C) reproduced with the permission from (Hinneburg et al. 2016).