Heparin/heparan sulfate analysis by covalently modified reverse polarity capillary zone electrophoresis-mass spectrometry

Abstract

Reverse polarity capillary zone electrophoresis coupled to negative ion mode mass spectrometry (CZE-MS) is shown to be an effective and sensitive tool for the analysis of glycosaminoglycan mixtures. Covalent modification of the inner wall of the separation capillary with neutral or cationic reagents produces a stable and durable surface that provides reproducible separations. By combining CZE-MS with a cation-coated capillary and a sheath flow interface, a rapid and reliable method has been developed for the analysis of sulfated oligosaccharides from dp4 to dp12. Several different mixtures have been separated and detected by mass spectrometry. The mixtures were selected to test the capability of this approach to resolve subtle differences in structure, such as sulfation position and epimeric variation of the uronic acid. The system was applied to a complex mixture of heparin/heparan sulfate oligosaccharides varying in chain length from dp3 to dp12 and more than 80 molecular compositions were identified by accurate mass measurement.

Keywords: Capillary zone electrophoresis; Covalent capillary coating; Mass spectrometry; Mixture analysis; Reverse polarity; Sulfated glycosaminoglycan.

Figure 1

Diagram depicting the forces of electroosmotic flow (EOF) and electrophoretic forces (EF) that act on analytes during a CZE-MS experiment with three different inner capillary surfaces: (A) Bare fused silica (BFS), (B) DMS coated, and (C) AHS coated.

Figure 2

Short term durability of neutral (DMS, solid line) and cation (AHS, dashed line; BSA, circle dashed line) coated capillaries shown using sample 1 across 150 iterations. The BSA trial was terminated after 81 iterations due to coating failure.

Figure 3

Electropherogram comparison of migration times based on different capillary coatings. Samples (1), (2), and (3) are tetrasaccharides with different numbers of sulfates. Sample (1) contains six sulfates, (2) has four sulfates, and (3) has three sulfates. Significant improvement in migration time and peak width is observed with neutral (DMS) and cation coated (AHS) capillaries.

Figure 4

A) Baseline separation of tetrasaccharide mixture containing samples 4 and 2 with different amino modifications. Mass spectrum of sample 4 (B) and 2 (C) showing the mass difference due to amino modification.

Figure 5

(A) Baseline CZE separation of a tetrasaccharide mixture on AHS capillary. Sample 5 and 6 are isomers with the same number of sulfate groups and exact mass, but differ in sulfate position on the last two sugar residues. (B) Mass spectrum of sample 5 demonstrating the observed charge state distribution.

Figure 6

Baseline separation of a stereoisomer mixture (samples 7 and 8) on AHS capillary. Sample 7 migrates first followed by sample 8. The shoulder peak labeled 7a is attributed to an anomeric form of sample 7.

Figure 7

A) Mass spectrum of Enoxaparin (LMWH) without separation. B) Separation windows of Enoxaparin on uncoated and coated capillaries: (1) BFS (2) DMS, and (3) AHS with migration times decreasing from 1 to 3. The presented migration time window varies between panels 1–3 to enable comparison.

Figure 8

Effect of additives on four tetrasaccharide standards with an increasing number of sulfates (samples 1–3 and 6) through an AHS coated capillary. The pH of BGE in AHS coated capillaries modulates migration time. Lower pH, from addition of formic acid, leads to faster migration, and higher pH, from diethylamine, leads to slower migration.