Heparan sulfates (HS) are linear, acidic polysaccharides belonging to the glycosaminoglycan (GAG) family. Each mature HS chain is biosynthesized as a polysaccharide of repeating disaccharide units (4GlcAβ1-4GlcNAcα1-) that undergoes a series of subsequent modification reactions.1
Because these reactions are not template driven, mature HS chains are extremely polydisperse, with varying sulfation patterns, acetylation compositions, epimerization sites and chain lengths.2
This heterogeneity and the essential roles HS plays in physiology drive the need for effective methods for correlation of HS structure and function.3
Detailed structural information on HS is needed to for basic biomedical research, development of therapeutics, and biomarker discovery efforts. Such efforts include chemical or chemoenzymatic synthesis of HS oligosaccharides4–7
, generation of structural libraries,8
metabolic engineering of HS biosynthesis9
and biomarker discovery.10
For each of these applications, it is necessary to correlate structure with biological function in the context of the heterogeneity that characterizes the HS compound class. As a result, it is desirable to develop methods to produce as much structural information as possible in a high throughput manner. Such analytical data will inform purification efforts appropriate with the goals of the project in question.
In previous work, we used HILIC LC-MS to profile compositions of HS oligosaccharides and other GAGs as part of comparative glycomics experiments.11–17
Recently, chip-based HILIC LC-MS with a post-column make-up flow (MUF) has shown great utility for the profiling of HS oligosaccharides.11, 12
The integration of a make-up flow on the HPLC-chip improved the spray stability generated from HILIC mobile phase over the course of a gradient elution for the separation of HS oligosaccharides. In addition, using organic solvent as the make-up solution enables use of a single spray voltage that is lower than that required to achieve stable spray for a mobile phase of high aqueous content during the entire HILIC gradient. This results in the enhanced capability of the HILIC LC-MS system to analyze longer HS oligosaccharides that elute at high aqueous gradient where electrospray would otherwise be unstable. This increased stability enables glycomics experiments requiring extended periods of stable instrument performance.
While the LC-MS compositional profiling information of HS oligosaccharides is useful, there is a clear need to extend this capability to the tandem MS dimension. Electron detachment dissociation (EDD) shows great potential for determination of fine structure of purified HS and other GAG oligosaccharides using static infusion.18–20
The technique requires several minutes of spectral acquisition and is most appropriate for detailed analysis of purified oligosaccharides, rather than high throughput structural analysis. Negative ion electron transfer dissociation (NETD) has been demonstrated to produce similar product ion patterns on GAG oligosaccharides as EDD with similar extended spectral acquisition times.21
Another option for tandem MS is to permethylate the oligosaccharides,22, 23
followed by chemical desulfation and acetylation of the formerly sulfated sites.24
The advantage of this approach, as demonstrated for chondroitin sulfate oligosaccharides, is that the derivatized products are stable and compatible with reversed phase LC/MS. The derivatization steps result in formation of some degree of side products, and it may be that this approach is also most appropriate for purified oligosaccharides.
HILIC chromatography has the advantage that it is directly compatible with tandem MS of native GAG oligosaccharides. HILIC LC-MS/MS is sensitive relative to EDD/NETD methods and does not require derivatization and workup. It is possible to use this approach to acquire tandem mass spectra in high throughput. Unfortunately, tandem MS of HS oligosaccharide precursor ions produces a mixture of product ions resulting from competing backbone dissociation versus those from losses of SO3
, and this is particularly true when using collision induced dissociation.25–29
This competition favors backbone cleavages as negative ion charge state increases (in absolute value) and as metal cation adducts are included in the precursor ions. The use of cations to facilitate tandem MS of saccharides has been appreciated since the early 1990s.30–34
Their application to LC-MS/MS experiments is limited by their potential to coat the source optics, cause loss of robust instrument performance and to increase mass spectral complexity.
Sulfolane and other reagents with low solution phase basicities and volatilities have been shown to increase the charge state of protein and protein complexes when added to the electrospray solution and have been utilized to facilitate top-down MS analysis in the positive ionization mode.35, 36
On-line LC/MS requires continuous infusion of spray solution, and there is concern that such non-volatile and highly viscous molecules will coat the MS ion source and optics and negatively impact the instrument response over time. Effective use of either type of additive would be facilitated by the ability to target the post-column addition precisely during the LC-MS runs.
The present work describes use of a chip-based system for automated pulsing of additives to the LC effluent to maximize backbone fragmentation of HS oligosaccharides in tandem MS. This system features software-controlled pulsing of additives at specified times during the LC elution. Using this system, the efficacies of a series of metal cations for pairing with HS oligosaccharides and improving the quality of the on-line tandem mass spectra were evaluated. The utility of sulfolane to supercharge negative ions during LC-MS analyses was also shown using the pulsed MUF chip. The pulsed MUF chip represents a means of introducing additives to LC effluents in a manner that improves the value of HS tandem mass spectra and minimizes quantity of non-volatile additives that enter the ion source.