Glycoform quantification of chondroitin/dermatan sulfate using a liquid chromatography-tandem mass spectrometry platform

Abstract

Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating uronic acid, N-acetylgalactosamine disaccharide units {[HexAbeta/alpha(1-3)GalNAcbeta(1-4)](n)()}. CS chains are polydisperse with respect to chain length, sulfate content, and glucuronic acid epimerization content, resulting in a distribution of glycoforms for a chain bound to any given serine residue. Usually, CS glycoforms exist, differing in sulfation position and uronic acid epimerization. This work introduces a novel LC-MS/MS platform for the quantification of mixtures of CS oligosaccharides. The CS polysaccharides were partially depolymerized and labeled with either the light (d(0)) or heavy (d(4)) form of 2-anthranilic acid (2-AA). Excess reagent was removed, and mixtures of the CS standard (d(0)) and unknown (d(4)) were made. The CS mixture was subjected to size exclusion chromatography (SEC) with on-line electrospray ionization mass spectrometric detection in the negative ion mode. Tandem mass spectra were acquired, and quantification of unknown samples within the mixture was made using relative ion abundances of specific diagnostic ions. The high accuracy and precision of the glycomics platform were demonstrated using glycoform mixtures made from standard CS preparations. The CS glycoform analysis method was then applied to cartilage extract, versican, and several dermatan sulfate preparations. This work presents the first application of a glycomics platform for the quantification of CS oligosaccharide mixtures for obtaining specific information about the positions of GalNAc sulfation and uronic acid epimerization.

Figure 1

The protocol for the LC/MS/MS platform presented.

Figure 2

A. Tandem mass spectrum of CSA d₀-2AA-Δdp4. B. Tandem mass spectrum of CSB d₄-2AA-Δdp4. C. Tandem mass spectrum of CSC d₄-2AA-Δdp4. D. Bar graph representation of the contribution of each diagnostic ion in the pure CS standards. CSA (white), CSB (gray), and CSC (dark gray). E. Structure of a CS tetrasaccharide and its diagnostic fragment ions. The Uronic Acid (UroA) and N-acetylgalactosamine (GalNAc) sulfate are symbolized by the open diamond and the open square, respectively.

Figure 3

A. Total Ion Chromatogram of a 10 μg binary 1:1 mixture of d₀-2AA-CSA: d₄-2AA-CSC. B. Tandem mass spectrum of the tetramer mixture of 1:1 d₀-2AA-CSA: d₄-2AA-CSC. Diagnostic ions (Y₁¹⁻, Y₃²⁻, and [M-H-SO₃]²⁻) are paired by 2 or 4 u depending on whether they are doubly or singly charged, respectively. C. Expanded mass range of the diagnostic ions. CSA labeled with the light form (d₀) of 2-AA was incorporated as an internal standard. In the case of the CS standard mixture above, the CSA standard sample labeled with d₀-2AA was mixed with a CSC sample (termed unknown) labeled with d₄-2AA.

Figure 4

Expanded mass regions for diagnostic ions of different CS samples. A. Tandem mass spectrum of a 10-μg tetramer ternary mixture of 1:1:1 d₀-2AA-CSA: d₄-2AA-CSB: d₄-2AA-CSC. B. Tandem mass spectrum of a 10-μg tetramer mixture of cartilage extract. Heavy (d₄) diagnostic ions used for quantification include: Y₁¹⁻ at m/z 425.1, Y₃²⁻ at m/z 441.5 and [M-H-SO₃]²⁻ at m/z 480.6. C. Tandem mass spectrum of a 10-μg tetramer mixture of Versican. CSA labeled with the light form (d₀) of 2-AA was incorporated as an internal standard. In the case of the CS standard mixture (A), the CSA standard samples labeled with d₀-2AA were mixed with CSB and/or CSC samples (termed unknown) labeled with d₄-2AA in varying ratios. In the case of proteoglycan samples (B and C), 3 μg of the CSA-d₀-2AA internal standard was added to each sample prior to LC/MS analysis. The CSA-d₀-2AA internal standard is represented by the peak at m/z 421.1.

Figure 5

A. Tandem mass spectrum of a 10-μg tetramer mixture of DS-18. Heavy (d₄) diagnostic ions used for quantification include: Y₁¹⁻ at m/z 425.0, Y₃²⁻ at m/z 441.5 and [M-H-SO₃]²⁻ at m/z 480.5. B. Tandem mass spectrum of a 10-μg tetramer mixture of DS-36. C. Tandem mass spectrum of a 10-μg tetramer mixture of DS-50. CSA labeled with the light form (d₀) of 2-AA was incorporated as an internal standard. The CSA-d₀-2AA internal standard (3 μg) was added to each sample prior to LC/MS analysis. The CSA-d₀-2AA internal standard is represented by the peak at m/z 421.1.