Analysis of heparin oligosaccharides by capillary electrophoresis–negative-ion electrospray ionization mass spectrometry

Abstract

Most hyphenated analytical approaches that rely on liquid chromatography-MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultra low molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE-MS coupling eletrospray ion source. Reverse polarity CE separation and negative mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The on-line CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis, bottom-up and top-down analysis of low molecular weight heparin. The application of this CE-MS method to ultra low molecular heparin suggest that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful on-line tandem MS analysis of heparins.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…In this regard, Lin et al. developed a CE‐MS method (negative ion mode) for the characterization of heparin/heparan sulfate derived disaccharides . In addition, Sanderson et al.…”

On‐line capillary electrophoresis/laser‐induced fluorescence/mass spectrometry analysis of glycans labeled with Teal™ fluorescent dye using an electrokinetic sheath liquid pump‐based nanospray ion source

“…Although high reactivity and separation efficiency of the Teal™ dye in CE/LIF assays have been reported earlier, MS detection of Teal‐labeled glycans is highly desirable to generate accurate identification and minimize peak assignment ambiguity. The CE/MS method that we adopted for the analysis of Teal‐labeled glycans uses an electrokinetically pumped nanospray sheath liquid CE/MS interface design (Figure S1, supporting information) . The analytes are injected into the separation capillary from the capillary inlet, which is placed inside the CE system.…”

“…When analytes migrate out of the capillary, the surrounding sheath liquid entrains the molecules and brings them into the mass spectrometer. We have demonstrated in earlier studies the use of this nanoflow sheath liquid CE/MS ion source for both positive and negative ion mode MS analysis of highly sulfated oligosaccharides …”

“…However, soon it was realized that the BGE of 0.7 M ammonia, 0.1 M ɛ‐aminocaprioic acid, 70% methanol as previously reported generated very high mass spectrometry background, preventing effective glycan identification. Taking into consideration that the previously reported CE/MS work was carried out using a much higher sheath liquid flow than that on the EMASS‐II ion source, it was decided to remove ɛ‐aminocaprioic acid from the formulation, and only use ammonium hydroxide and methanol in the BGE buffer. Figure shows the CE/MS result of a mixture of Teal‐A2F and unlabeled A2F using 0.5 M ammonium hydroxide, 50% methanol.…”

Recent Advances in the Analysis of Complex Glycoproteins

“…Chem. 2016 , DOI: 10.1007/s00216-016-9662-1 [June 20 Epub ahead of Print] (ref 204). Copyright 2016 Springer.…”