MUCINS: A BLACK BOX OF BIOLOGY
The overarching goal of the Malaker Lab is to develop methods that allow for mass spectrometry analysis of mucins, which are densely O-glycosylated proteins. Understanding mucin structure and site-specific glycosylation will allow us to unravel the complex relationship between mucins and human disease. Projects are broadly broken down into three main areas:
The dense O-glycosylation in mucin domains makes these proteins resistant to digestion by workhorse proteases such as trypsin, and only recently did our work characterize the ability of bacterial “mucinases” to proteolytically digest endogenous mucins. As these enzymes are useful in glycoproteomic workflows, staining procedures, and enrichment of mucins, my laboratory will mine the gut microbiota for mucinases that can be used for these purposes. We will then characterize their cleavage motifs using a combination of biochemistry and mass spec techniques. We will also investigate their endogenous biological functions.
One of the most problematic aspects of studying mucins by MS is that the ionization efficiency of glycopeptides is significantly lower than their unmodified counterparts. During the electrospray ionization (ESI) process, nanodroplets are formed when the charge at the surface of the droplet creates enough electrostatic repulsion to exceed the surface tension of the solvent droplet. Polar species, such as densely glycosylated peptides, migrate to the droplet interior to optimize solvation energy. Hydrophobic species, such as unmodified peptides, are ejected from the droplets and funneled into the inlet of the mass spectrometer. The hydrophilic species, on the other hand, weakly ionize, likely remaining within the droplet of solvent or are neutralized near the inlet of the mass spectrometer. Thus, in order to be able to detect highly glycosylated domains of mucins, this process must be altered to accommodate hydrophilic species.
Several search algorithms are available for reliable identification of unmodified peptides, but all of them fall short when glycosylation is added as a variable modification. Several problems arise when searching for glycosylation, namely: (a) poor fragmentation of the glycopeptides, (b) large number of possible glycans, and (c) utilization of collision-induced spectra for site-localization. In my laboratory, we will design in-house software that can address these issues and confidently assign glycopeptides. The program will extract collision-induced fragmentation spectra and use these spectra to determine the glycan structure and naked peptide sequence. Then, paired radical electron-induced fragmentation spectra will be used to site-localize the glycan on the peptide backbone.