Glycomic remodeling of glycocalyx and mucus

Mucins are large, highly O- and partially N-glycosylated proteins that form the polymer matrix of the mucus. The protein backbone of mucins is relatively well characterized, even though the sequence coverage in classical proteomics experiments is typically low (20-30%) with a bias for non-glycosylated protein regions. However, very little is known about the structures and dynamics of mucus glycosylation and the resulting consequences on the macroscopic behavior. This is largely a result of three fundamental challenges: 1) the highly complex structure of glycans with the frequent coexistence of multiple isomers, 2) the site heterogeneity with multiple glycosylated positions along the protein sequence and 3) the dense glycosylation, which prevents an efficient enzymatic digestion into glycopeptides for sequencing. Here we will address these problems using proteomics, glycomics and glycoproteomics techniques and a combination of isotope labeling, partial chemical glycan release and proteolysis using different enzymes. First, we will extract and purify hydrogel glycoproteins from different sources and compare their proteomics profiles. The purified proteins will then be made available to other groups within the CRC. Second, the global glycosylation profile of the investigated proteins will be determined by analyzing the chemically released glycans using mass spectrometry (MS) and ion mobility-mass spectrometry (IM-MS). Third, new approaches will be developed to generate intact glycopeptides from hydrogel proteins by partial chemical deglycosylation using mild acids or glycosidases and subsequent proteolytic digestion. Finally, the so-obtained intact glycopeptides will be analyzed using isotope labeling, IM-MS and novel ultraviolet photodissociation MS (UVPD-MS). All developed analytical approaches will be transferred to the service project Z01 and made available to other groups within the CRC.