Hydrophilic interaction anion exchange for separation of multiply modified neutral and anionic Dictyostelium N-glycans

Abstract

The unusual nature of the N-glycans of the cellular slime mould Dictyostelium discoideum has been revealed by a number of studies, primarily based on examination of radiolabeled glycopeptides but more recently also by MS. The complexity of the N-glycomes of even glycosylation mutants is compounded by the occurrence of anionic modifications, which also present an analytical challenge. In this study, we have employed hydrophilic interaction anion exchange (HIAX) HPLC in combination with MALDI-TOF MS/MS to explore the anionic N-glycome of the M31 (modA) strain, which lacks endoplasmic reticulum α-glucosidase II, an enzyme conserved in most eukaryotes including Homo sapiens. Prefractionation with HIAX chromatography enabled the identification of N-glycans with unusual oligo-α1,2-mannose extensions as well as others with up to four anionic modifications. Due to the use of hydrofluoric acid treatment, we were able to discriminate isobaric glycans differing in the presence of sulphate or phosphate on intersected structures as opposed to those carrying GlcNAc-phosphodiesters. The latter represent biosynthetic intermediates during the pathway leading to formation of the methylphosphorylated mannose epitope, which may have a similar function in intracellular targeting of hydrolases as the mannose-6-phosphate modification of lysosomal enzymes in mammals. In conclusion, HIAX in combination with MS is a highly sensitive approach for both fine separation and definition of neutral and anionic N-glycan structures.

Keywords: Glycome; HPLC; Mass spectrometry; Phosphate; Sulphate.

Figure 1. Summary of biosynthesis of N-glycans in Dictyostelium, relevant diagnostic MALDI-TOF-MS/MS fragments and HIAX-HPLC.

(A) Simplified ‘wild-type’ biosynthetic scheme starting with Glc₃Man₉GlcNAc₂ showing the two-step action of glucosidase II (absent in the M31 strain) followed by the action of GlcNAc-1-phosphotransferase (GPT), removal of the GlcNAc and methylation of the ‘uncovered’ phosphate by a methyltransferase (MeT); the linkages, arms (A, B and C) and locations of intersecting and bisecting GlcNAc are also annotated. (B) Summary of important diagnostic ions of single and multiple anionic N-glycans obtained from MS/MS in negative ion mode (positive ion mode m/z values of phosphate-containing fragment ions are also indicated with (+)); multiple sulphated ions were detected as sodium or potassium adducts (Na and/or K, shown in brackets). (C) HIAX chromatograms of the neutral and anionic pools highlighting the elution positions for three isobaric structures of m/z 2566.

Figure 2. Mannosidase digestion of selected glycans with single and multiple anionic modifications.

HIAX-fractionated singly-modified Hex₉HexNAc_2-3S₁ or Hex₉HexNAc_2-3P₁Me_0-1 N-glycan structures (A-F) and those with multiple sulphate and methylphosphate modifications (G-L) are shown before and after jack bean α-mannosidase digest in negative ion mode. Hex_8,10-11HexNAc_2-4-based sulphated or methylphosphorylated structures which co-elute with those based on Hex₉HexNAc₂ are shown in a greyscale; asterisks in panel A indicate contaminants of m/z 2154 and 2220. The parent structures, but not the in source fragment ions, are annotated with the composition (as H_8-11N_2-4P_0-1Me_0-1S_0-3; i.e., Hex_8-11HexNAc_2-4P_0-1Me_0-1S_0-3); in source losses of sulphate are indicated by ∆ 102 or 118 (i.e., loss of sulphate and sodium or potassium counterions), whereas loss of mannose residues upon enzymatic digestion are indicated by red or blue dashed lines. Increasing numbers of anionic modifications as well as the presence of the intersecting GlcNAc residue result in decreased susceptibility to mannosidase digestion (a shift towards sodium adducts was observed due to the composition of the buffer in which the mannosidase is supplied). MS/MS for selected Hex₉-based structures have been previously published ; further example digests as well as ‘zoomed-in’ regions of panels I and K (showing annotations of all adducts) are shown in Supplementary Figure 2 and selected MS/MS of mannosidase digestion products are in Supplementary Figure 3 A-D. Based on analyses of an RP-HPLC fraction containing Man_8-9GlcNAc₂PMe (panel C), it is concluded that PMe is on the C-arm (see also Supplementary Figure 4). Further data on the m/z 2566 glycans (panels E and G) are also presented in Supplementary Figure 5.

Figure 3. Analysis of isobaric anionic N-glycans found in the M31 glucosidase II mutant strain.

N-glycan structures with the monoisotopic m/z 2566 as [M-H]^- were identified in three different RP-HPLC fractions (A, D and H). In the case of the 7.8 g.u. structure (m/z 2566; A), resistance to both shrimp alkaline phosphatase (SAP; B) and hydrofluoric acid (HF; C) were compatible with it being sulphated (compare with Supplementary Figure 5 A and B). The 5.0 g.u. fraction contained two major glycans detected in both negative and positive modes (m/z 2256/2258 and 2566/2569; D and E), both of which were sensitive to hydrofluoric acid (G; loss of 94 [PMe] or 283 Da [HexNAcP], with products only detectable in positive mode) but resistant to phosphatase (F); thus the Hex₁₁HexNAc₃P glycan possesses a HexNAc-1-P phosphodiester (see also Supplementary Figure 5 E-G). The 5.6 g.u. fraction contained two major glycans (m/z 2457 and 2566; H), both of which are sensitive to hydrofluoric acid (K and L; loss of 94 [PMe] or 80 [P] Da); in this case, the Hex₁₁HexNAc₃P glycan possesses a phosphatase-sensitive phosphomonoester (see also Supplementary Figure 5 C and D), while the methylated phosphate on Hex₁₁HexNAc₂PMe is phosphatase-resistant. For MS/MS of the corresponding m/z 2566 glycans isolated from HIAX fractions as well as digestion products, see Supplementary Figure 5 H-L.

Figure 4. Identification of a novel oligo-α1,2-mannose modification on neutral N-glycans.

(A-C) MALDI-TOF MS of a late-eluting neutral HIAX fraction of M31 N-glycans (see also chromatogram in Figure 1C) before and after digestion with Aspergillus α1,2-specific mannosidase alone or, after heat inactivation, subsequently with an insect cell α-glucosidase II; corresponding MS/MS are shown in Supplementary Figure 6. (D-F) MALDI-TOF MS of another late-eluting neutral HIAX fraction before and after digestion with Aspergillus α1,2-specific mannosidase or bacterial endo-α-mannosidase; corresponding MS/MS are shown in Supplementary Figure 7. Losses of hexose residues are indicated by red or blue dashed lines, key Y-fragments by numbers in square brackets and contaminating ions with an asterisk. (G-J) The RP-HPLC elution time and MS/MS fragmentation pattern of the endo-α-mannosidase product (panel F and Supplementary Figure 7C) was compared to known bisected (isomer I) and intersected (isomer II) wild-type AX3 Hex₈HexNAc₃ glycans; on this basis, it was concluded that the Hex₁₃HexNAc₃ glycan containing the oligo-α1,2-mannose extension (panel D) contains an intersecting GlcNAc residue.

Figure 5. Summary of the anionic modifications found in the M31 glucosidase II mutant strain.

Schematic overview of Glc_0-2Man_8-9GlcNAc_2-3P_0-1PMe_0-2S_0-3 structures based on their HIAX-HPLC elution properties; the numbers of anionic modifications (one, two or more; i.e., R_1-4) are indicated by the grading of the shaded regions (with the lightest background for singly-modified glycans). The annotation of the structures was based on MALDI-TOF-MS/MS analysis in positive and negative ion modes combined with glycosidase and chemical treatments.