Speakers
Description
INTRODUCTION: Conventional Mass Spectrometry (MS) techniques detect gas-phase ions based on their mass-to-charge ratio (m/z), whereas Ion Mobility-MS instruments additionally separate ions according to their size and shape. This orthogonal technique is particularly adapted for challenging samples such as glycoconjugates, which often present multiple isomers due to their inherent complex structures. Ions are typically guided through the mobility cell, which is filled with a buffer gas, by a weak electric field. As they go through the IMS cell, ions collide with the buffer gas. Extended structures encounter more collisions than compact molecules, and thus traverse the cell with a lower velocity. Isomeric species that exhibit the same m/z-ratio can then be separated by their distinct shape. The measured drift time can be used to estimate the collision cross section (CCS), which is a property intrinsic to the molecule that can help structural identifications using databases, molecular standards, or calculated models.
TRAPPED ION MOBILITY: The timsTOF Pro (Bruker), one of the new generation IM-MS instruments, uses Trapped Ion Mobility Spectrometry (TIMS) to separate ions. Here, the ions are dragged into the spectrometer by a nitrogen gas flow, while an electric field in the opposite direction is used to decelerate the ions. This allows trapping of the ions and subsequent release, depending on their CCS, by gradually decreasing the electric field. TIMS exhibits an increased resolution during the IM separation, an improved duty cycle, and enables a softer transit of the ions into the spectrometer. Here, the TIMS separation is applied to the analysis of two types of glycoconjugates, glycosaminoglycans (GAGs) and O-glycans, which are ionized using an in-house 3D-printed offline nanoESI source connected to the entrance of the timsTOF instrument.
GAGS APPLICATION: The high-resolution instrument is used to develop an IM-MS based GAG disaccharide analysis strategy. Already GAG disaccharides posses a high amount of isomeric species and are therefore not distinguishable by MS methods alone. Conventional condensed-phase techniques such as HPLC are limited in their sample consumption and needed analysis time. IM-MS can help reduce both by a ten-fold and therefore increase sample throughput while at the same time decrease produced chemical waste.
O-GLYCOMICS APPLICATION: Deprotonated O-glycans are separated in the TIMS cell and characterized by CID. A new IM-MS/MS workflow is developed using O-glycans released from mucins. The high-resolution ion mobility of the TIMS allows to separate the O-glycan isomers and even to detect new structures. The workflow is successfully applied to clinical sputum samples and characteristic glycosylation features are measured and compared between the healthy and Cystic Fibrosis patients. Compared to the traditionally employed LC-MS/MS methods, the analysis time is drastically reduced, to acquisitions of few minutes. As a high throughput solution, TIMS coupled to MS constitutes a promising alternative for O-glycomics.
