Integrating High-Resolution Cyclic Ion Mobility Separations with Tandem Mass Spectrometry and Collision Cross Section Measurements for Human Milk Oligosaccharide Sequencing

Human milk oligosaccharides (HMOs) are a biologically important class of carbohydrates responsible for promoting the healthy development of infants. However, to better understand their specific biological roles, analytical techniques are needed to unambiguously characterize them. While liquid chromatography–tandem mass spectrometry (LC-MS/MS) remains the gold standard for HMO analysis, new orthogonal techniques are desired for improving their isomer analysis. Ion mobility spectrometry–mass spectrometry (IMS-MS) has emerged as a complementary technique to LC-MS/MS but has seen little use toward HMO sequencing analysis beyond the construction of collision cross section (CCS) databases. In this work, we describe the use of collision-induced dissociation performed prior to high-resolution cyclic ion mobility separations (i.e., pre-cIMS CID) in conjunction with CCS measurements to characterize the linkage positioning in various HMOs irrespective of the starting precursor ion. We then demonstrated how our developed approach could be used to sequence an unknown HMO present in a purified extract. Lastly, we applied our workflow to sequence an isomeric mixture in the same extract using cIMS/cIMS instead of pre-cIMS CID. Overall, our developed approach is a first step toward standard-free de novo HMO sequencing as well as being a complementary and orthogonal method to existing LC-MS/MS-based workflows.