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.