Positive Mode LC-MS/MS Analysis of Chondroitin Sulfate Modified Glycopeptides Derived from Light and Heavy Chains of The Human Inter-α-Trypsin Inhibitor Complex

Abstract

The inter-α-trypsin inhibitor complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The inter-α-trypsin inhibitor complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcAβ3Galβ3Galβ4Xylβ-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the nonsulfated nonreducing end, (GalNAcβ4GlcAβ3)(n), to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation. The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal GalNAc residues. The fragmentation behavior of CS glycopeptides under variable higher-energy collisional dissociation conditions displays an energy dependence that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.

Fig. 1.

Schematic depiction of the inter-α-trypsin inhibitor complex and its degradation by chondroitinase ABC. Both the linkage and cross-linkage regions are highlighted as well as the molecular details of the GalNAc to aspartic acid ester bond that cross-links the heavy chains to the CS chain of bikunin. In addition to CS-glycopeptides the endolytic activity of the chondroitinase ABC digestion is also expected to generate free disaccharides of the CS chain (faded region). The formation of unsaturated GlcA by chondrotinase ABC is shown below the complex and the monosaccharide symbols are explained in the upper left quadrant.

Fig. 2.

Total ion chromatograms of SAX-enriched CS-glycopeptides from a urine sample. Three fractions collected at (A) 400 mm, (B) 800 mm, and (C) 1600 mm NaCl were subjected to chondroitinase ABC hydrolysis and analyzed with nLC-MS/MS.

Fig. 3.

MS/MS fragmentation patterns recorded at different HCD levels from a triple charged bikunin CS-glycopeptide precursor ion at m/z 1094.44. (A) HCD performed at normalized collision energy of 20%; and (B) of 30%. Glycopeptide fragments as well as b- and y-ions are annotated. The schematic structure of the precursor ion is shown (boxed structure).

Fig. 4.

MS/MS fragmentation patterns recorded at the 20% HCD level from a sodiated bikunin CS-glycopeptide precursor ion, [M+3Na]³⁺ at m/z 1116.41. A, The complete HCD spectrum. B, the m/z 180–370 region; C, the m/z 1000–1500 region; and D, the m/z 900–1000 region. All major ions are annotated to charge, m/z, Na⁺ content and glycan structure.

Fig. 5.

MS/MS fragmentation patterns recorded at the 20% HCD level from a disulfated and sialylated bikunin CS-glycopeptide precursor ion. A, The complete HCD spectrum of the [M+3H]³⁺ precursor ion at m/z 1191.46. B, The complete HCD spectrum of the [M+3Na]³⁺ precursor ion at m/z 1213.44 and (C) the expanded m/z 1000–1500 region of the sodiated precursor ion.

Fig. 6.

MS/MS fragmentation patterns recorded at the 20% HCD level from a disulfated and fucosylated bikunin CS-glycopeptide precursor ion. A, The complete HCD spectrum of the [M+3H]³⁺ precursor ion at m/z 1143.11. B, The complete HCD spectrum of the [M+3Na]³⁺ precursor ion at m/z 1165.09, and C, the expanded m/z 1000–1500 region of the sodiated precursor ion.

Fig. 7.

Total ion chromatograms of three fractions of a SAX purified and chondroitinase ABC treated urine sample with all major peaks annotated as different glycoforms of bikunin. The first fraction was eluted with 400 mm NaCl (top), the second fraction with 800 mm NaCl (middle), and the last fraction with 1600 mm NaCl (bottom).

Fig. 8.

MS/MS fragmentation spectra of H1 and H1+H2 Heavy chain peptides cross-linked to CS-chains hydrolyzed by chondroitinase ABC. The H1 C-terminal tryptic and semitryptic peptides LPDRVTGVDTD and VTGVDTD were identified cross-linked to ΔGlcAGalNAc at m/z 774.85 (A), to ΔGlcAGalNAcGlcAGalNAc at m/z 964.41 (B), to ΔGlcAGalNAcGlcAGalNAc at m/z 723.77 (C) and together with the C-terminal peptide VEND of H2 cross-linked to ΔGlcAGalNAcGlcAGalNAc at m/z 952.36 (D) and cross-linked to ΔGlcAGalNAcGlcAGalNAcGlcAGalNAc at m/z 1142.42 (E). Schematic structures of the corresponding precursor ions are shown in the box.