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Complex carbohydrates are ubiquitous in nature and represent one of the major classes of biopolymers. They can exhibit highly diverse structures with multiple branched sites as well as a complex regio- and stereochemistry. A common way to analytically address this complexity is liquid chromatography (LC) in combination with mass spectrometry (MS). However, MS-based detection often does not provide sufficient information to distinguish glycan isomers. Ion mobility-mass spectrometry (IM-MS)─a technique that separates ions based on their size, charge, and shape─has recently shown great potential to solve this problem by identifying characteristic isomeric glycan features such as the sialylation and fucosylation pattern. However, while both LC-MS and IM-MS have clearly proven their individual capabilities for glycan analysis, attempts to combine both methods into a consistent workflow are lacking. Here, we close this gap and combine hydrophilic interaction liquid chromatography (HILIC) with IM-MS to analyze the glycan structures released from human alpha-1-acid glycoprotein (hAGP). HILIC separates the crude mixture of highly sialylated multi-antennary glycans, MS provides information on glycan composition, and IMS is used to distinguish and quantify α2,6- and α2,3-linked sialic acid isomers based on characteristic fragments. Further, the technique can support the assignment of antenna fucosylation. This feature mapping can confidently assign glycan isomers with multiple sialic acids within one LC-IM-MS run and is fully compatible with existing workflows for N-glycan analysis.

Mass spectrometry is routinely employed for structure elucidation of molecules. Structural information can be retrieved from intact molecular ions by fragmentation; however, the interpretation of fragment spectra is often hampered by poor understanding of the underlying dissociation mechanisms. For example, neutral headgroup loss from protonated glycerolipids has been postulated to proceed via an intramolecular ring closure but the mechanism and resulting ring size have never been experimentally confirmed. Here we use cryogenic gas-phase infrared (IR) spectroscopy in combination with computational chemistry to unravel the structures of fragment ions and thereby shed light on elusive dissociation mechanisms. Using the example of glycerolipid fragmentation, we study the formation of protonated five-membered dioxolane and six-membered dioxane rings and show that dioxolane rings are predominant throughout different glycerolipid classes and fragmentation channels. For comparison, pure dioxolane and dioxane ions were generated from tailor-made dehydroxyl derivatives inspired by natural 1,2- and 1,3-diacylglycerols and subsequently interrogated using IR spectroscopy. Furthermore, the cyclic structure of an intermediate fragment occurring in the phosphatidylcholine fragmentation pathway was spectroscopically confirmed. Overall, the results contribute substantially to the understanding of glycerolipid fragmentation and showcase the value of vibrational ion spectroscopy to mechanistically elucidate crucial fragmentation pathways in lipidomics.

The increasing antibiotic resistance in bacteria is an alarming phenomenon all around the world. Certain strains have developed resistance against multiple antimicrobial molecules, in which cases, the final option is to use a last-resort drug. These drugs, however, are last-resort for a reason: they can pose serious risk on vital organ functions in the patient. To mitigate the risk of severe side-effects and to reduce the rate of bacterial mutation, co-administration with other molecules that increase their efficacy seems to be the only suitable option. This leads to a reduced dose while maintaining the same level of antibiotic activity within the body. In this study, the effect of heparin derivatives on the antibiotic activity of colistin and their interactions were studied by ion mobility, mass spectrometry, and bacterium growth assays. The results show that during the association of colistin and heparin, they retain their structure while higher-stoichiometry complexes can form. When long-chain heparin is co-administered, multiple colistin molecules can associate with it, which increases the antibiotic activity by ∼40% relative to the sole administration of colistin.