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Despite evident regulatory roles of heparan sulfate (HS) saccharides in numerous biological processes, definitive information on the bioactive sequences of these polymers is lacking, with only a handful of natural structures sequenced to date. Here, we develop a “ S hotgun”  I on  M obility  M ass  S pectrometry  S equencing (SIMMS 2 ) method in which intact HS saccharides are dissociated in an ion mobility mass spectrometer and collision cross section values of fragments measured. Matching of data for intact and fragment ions against known values for 36 fully defined HS saccharide structures (from di- to decasaccharides) permits unambiguous sequence determination of validated standards and unknown natural saccharides, notably including variants with 3 O -sulfate groups. SIMMS 2  analysis of two fibroblast growth factor-inhibiting hexasaccharides identified from a HS oligosaccharide library screen demonstrates that the approach allows elucidation of structure-activity relationships. SIMMS 2  thus overcomes the bottleneck for decoding the informational content of functional HS motifs which is crucial for their future biomedical exploitation.

Shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides