U.S. Department of Energy

Pacific Northwest National Laboratory

Enhancing Carbohydrate Isomer Separation with Ion Mobility Spectrometry-Mass Spectrometry

Introduction 

Glycomics has become an important field of research since carbohydrates play a critical role in many biology events processes such as molecular recognition, signaling and cell communication. Carbohydrates also often conjugate with other biomolecules, such as proteins and lipids, and have significant effects on their properties of the molecules they attach. However, carbohydrates are difficult to characterize due to the variety and complexity of their  structural compositionstructures, types of glycosidic linkages, and their configurations. Here we use an ion mobility spectrometry-mass spectrometry (IMS-MS) platform to characterize analyze standard carbohydrate isomers and also characterize biologically relevant αGal-containing O-glycans that are important targets for protective antibodies against Trypanosoma cruzi, which causes Chagas disease. 
Methods 

Carbohydrate standards and complex glycans were analyzed with both a home-built IMS-QTOF MS and an Agilent 6560 IMS-QTOF MS platform using both positive and negative polarities to understand whether the detected carbohydrate isomers were separable. Once injected into theAfter nano ESI source, the carbohydrate ions were passed through the inlet capillary, focused by a high pressure electrodynamic ion funnel, and accumulated in a lower pressure ion funnel trap. The ions were then pulsed into the IMS drift cell and refocused by a second ion funnel at the drift cell exit prior to QTOF MS detection.

 
Preliminary Data 

Carbohydrate standards, including monosaccharides, disaccharides and polysaccharides, were analyzed with the IMS-QTOF-MS systemplatform. Interestingly, the IMS profiles for most carbohydrates in positive and negative ion modes are significantly different. For example, the monosaccharides xylose and arabinose showed similar drift times in negative mode and were not separable. However, in positive ion mode the sodiated xylose and arabinose were easily separated. In another case, ketose, a trisaccharide was shown to travel fasterhave much higher mobility than raffinose in positive mode, but slower lower in negative mode. Such IMS profile changes between positive and negative mode were observed in many cases through this study, however most group preferprevious work has analyzing theed carbohydrates in only the positive mode due to its the easier more efficient ionization nature. Our results illustrate that it using both positive and negative ion modes t is crucial to analyze the for carbohydrates using both positive and negative ion modes to often enables achieve achieving better separations. 
Carbohydrate isomers and complex synthetic αGal-containing O-glycan isomers consisting of slight structural differences such as α- versus β-linkages, 1-3 vs. 1-4 linkage connectivity, and linear vs. branched structures were also evaluated with the IMS-MS platform.  These slight changes are thought to have huge biological implications, so their separation is vital to understanding complex biological samples. In each case, the isomers were distinguished in either the positive or negative mode, however, not fully in some instances. To gain better separation, metal cations adducts were evaluated. We observed that divalent metals such as Mg2+ and Zn2+ were able to nicely separate some isomers that had very similar drift times with Na+ or when deprotonated. 

Novel Aspect 

The effect of charge polarity and metal cations on for distinguishing carbohydrate isomers with by IMS-MS and understanding biologically relevant structural changes.

 

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