Structural analysis of bikunin glycosaminoglycan

Abstract

The structure of an intact glycosaminoglycan (GAG) chain of the bikunin proteoglycan (PG) was analyzed using a combined top-down and bottom-up sequencing strategy. PGs are proteins with one or more linear, high-molecular weight, sulfated GAG polysaccharides O-linked to serine or threonine residues. GAGs are often responsible for the biological functions of PGs, and subtle variations in the GAG structure have pronounced physiological effects. Bikunin is a serine protease inhibitor found in human amniotic fluid, plasma, and urine. Bikunin is posttranslationally modified with a chondroitin sulfate (CS) chain, O-linked to a serine residue of the core protein. Recent studies have shown that the CS chain of bikunin plays an important role in the physiological and pathological functions of this PG. While no PG or GAG has yet been sequenced, bikunin, the least complex PG, offers a compelling target. Electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry (ESI FTICR-MS) permitted the identification of several major components in the GAG mixture having molecular masses in a range of 5505-7102 Da. This is the first report of a mass spectrum of an intact GAG component of a PG. FTICR-MS analysis of a size-uniform fraction of bikunin GAG mixture obtained by preparative polyacrylamide gel electrophoresis, allowed the determination of chain length and number of sulfo groups in the intact GAGs.

Figure 1

(A) Positive-ion, linear MALDI-TOF mass spectrum of bikunin PG. Calibrant peaks are labeled Lys (chicken egg lysozyme) and Ins (bovine insulin). (B) Negative-ion, linear MALDI-TOF mass spectrum of bikunin peptidoglycan. Unassigned peak is marked with an asterisk.

Figure 2

Outline of the analytical strategy, showing amounts of sample in micrograms used in each experimental step.

Figure 3

Improvement in S/N in the FTICR mass spectrum of the bikunin CS mixture (a) achieved by acquiring MS data over narrow m/z regions using quadrupole mass filter (b). Combining mass spectra acquired over narrow overlapping m/z regions afforded a full spectrum shown in panel (c).

Figure 4

Experimental (a) and simulated (b) negative-ion FTICR mass spectra of the bikunin CS-A mixture showing [M − 15H + 5Na]⁻¹⁰ ion which was assigned to GalNAc(GlcAGalNAc)₁₁(SO₃)₆GlcAGalGalXylol.

Figure 5

EDD mass spectrum of the NRE monosulfated trisaccharide obtained through the endolytic chondroitinase AC digestion of the bikunin CS.

Figure 6

(A) PAGE analysis of bikunin CS. Direction of migration is from top to bottom. Lane 1, bikunin CS mixture; lanes 2–8, fractions of the mixture isolated by preparative PAGE followed by electroblotting. (B) FTICR-MS analysis of the fraction in lane 6: portion of the spectrum showing peaks corresponding to several components with multiple charge states. (C) Experimental and simulated FTICR mass spectra of the 603.13 m/z peak, corresponding to [M – 11H]¹¹⁻ ion of a hexasulfated, 33-monosaccharide CS chain.