Structure Determination of Glycoproteins by Mass Spectrometry

The relevant background information about the glycoprotein, enzymatic digestion, and the new matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDITOF MS) ionization technique is reviewed. This method allows the detection of a complex mixture over a wide mass range (500-200,000 Da) using picomole to femtomole amounts of sample. Therefore, it is now possible to determine the structure of a glycoprotein using relatively little material and provide precise information about the glycosylation distribution. In this thesis, two glycoproteins were investigated by the combination of various enzyme digestions and MALDI-TOF MS. The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by MALDI MS. The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized. This information goes far beyond previously published data and sometimes differs from that. During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (A58++T, N65-*H, and V412++A). In addition, an unknown glycoprotein that is secreted by the human axilla, originally named apocrine secretion odor binding protein (ASOB2) was studied. This protein has a blocked N-terminus and thus is not suitable for Edman sequencing. The partial sequences of some peptides generated by Lys-C digestion were deduced from MALDI-post-source decay spectra. By individually matching the two most reliable sequences with the protein database, it was revealed that ASOB2 is a known glycoprotein, human apolipoprotein D (apo D). Detailed mass spectral data confirmed that the amino acid sequences are completely identical. However, the type of glycosylation of axillary apo D was found to be different from that reported recently for human plasma apo D. It was also shown that the odor producing component, 3-methyl-2hexenoic acid, carried by this protein is not covalently bound. Thesis supervisor: Klaus Biemann Title: Professor of Chemistry