IR spectroscopy of gas phase ions is proposed to resolve positional isomers of sulfated carbohydrates. Mass spectrometric fingerprints and gas phase vibrational spectra in the near and mid IR regions were obtained for sulfated monosaccharides, yielding unambiguous signatures of sulfated isomers. We report the first systematic exploration of the biologically relevant but notoriously
challenging deprotonated state in the near IR region. Remarkably, anions displayed very atypical vibrational profiles, which challenge the well-‐established
DFT (Density Functionnal Theory) modeling. The proposed approach was used
to elucidate the sulfate patterns in glycosaminoglycans – a ubiquitous class of mammalian carbohydrates – which is regarded as a major challenge in carbohydrate structural analysis. Isomeric glycosaminoglycan disaccharide from heparin and chondroitin sources where resolved, highlighting the potential of InfraRed Multiple Photon Dissociation spectroscopy as a novel structural tool for carbohydrates.