Dried blood spot N-glycome analysis by MALDI mass spectrometry

Body fluid N-glycome analysis as well as glyco-proteoform profiling of existing protein biomarkers potentially provides a stratification layer additional to quantitative, diagnostic protein levels. For clinical omics applications, the collection of a dried blood spot (DBS) is increasingly pursued as an alternative to sampling milliliters of peripheral blood. Here we evaluate DBS cards as a blood collection strategy for protein N-glycosylation analysis aiming for high-throughput clinical applications. A protocol for facile N-glycosylation profiling from DBS is developed that includes sialic acid linkage differentiation. This protocol is based on a previously established total plasma N-glycome mass spectrometry (MS) method, with adjustments for the analysis of DBS specimens. After DBS-punching and protein solubilization N-glycans are released, followed by chemical derivatization of sialic acids and MS-measurement of N-glycan profiles. With this method, more than 80 different glycan structures are identified from a DBS, with RSDs below 10% for the ten most abundant glycans. N-glycan profiles of finger-tip blood and venous blood are compared and short-term stability of DBS is demonstrated. This method for fast N-glycosylation profiling of DBS provides a minimally invasive alternative to conventional serum and plasma protein N-glycosylation workflows. With simplified blood sampling this DBS approach has vast potential for clinical glycomics applications.