Comparative analysis of INLIGHT™-labeled enzymatically depolymerized heparin by reverse-phase chromatography and...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Glycoscience

Background:

• Characterizing the microheterogeneity of glycosaminoglycans (GAGs) like heparin and heparan sulfate presents significant analytical challenges.
• Existing methods often struggle with detailed structural elucidation of these complex polysaccharides.
• High-resolution/accurate mass (HRAM) mass spectrometry coupled with liquid chromatography (LC-MS/MS) is a powerful tool for GAG analysis.

Purpose of the Study:

• To present a novel stable isotope-labeled hydrazide tag (INLIGHT™) for characterizing heparinase-digested heparin (digHep) products.
• To demonstrate the application of INLIGHT™ labeling with HRAM reverse-phase LC-MS/MS for detailed GAG structural analysis.
• To establish a foundation for quantitative comparative analyses of various heparin and heparan sulfate forms.

Main Methods:

• Utilized a stable isotope-labeled hydrazide tag (INLIGHT™) for reducing end labeling of heparin fragments.
• Employed high-resolution/accurate mass (HRAM) reverse-phase LC-MS/MS for separation and detection of labeled GAGs.
• Performed both full scan LC-MS and data-dependent LC-MS/MS analyses to identify and structurally characterize digHep species.

Main Results:

• Identified 116 unique heparinase-digested heparin (digHep) species using INLIGHT™ labeling and LC-MS/MS.
• Structurally elucidated 83 digHep products, including disaccharides, tetrasaccharides (DP4), hexasaccharides (DP6), octasaccharides (DP8), and decasaccharides (DP10).
• Confirmed identification confidence through co-elution of light and heavy INLIGHT™ tags for all identified digHep species.

Conclusions:

• The INLIGHT™ labeling strategy coupled with HRAM reverse-phase LC-MS/MS effectively overcomes analytical challenges in GAG structural characterization.
• This method allows for detailed mapping and identification of depolymerized heparin and heparan sulfate products without ion-pairing reagents.
• The approach provides a robust foundation for precise quantitative comparative analyses of diverse GAGs.