Nicolle Hannah Packer

Localization of O-Glycosylation Sites on Glycopeptide Fragments from Lactation-associated MUC1 ALL PUTATIVE SITES WITHIN THE TANDEM REPEAT ARE GLYCOSYLATION TARGETS IN VIVO

Since there is no consensus sequence directing the initial GalNAc incorporation into mucin peptides,O-glycosylation sites are not reliably predictable. We have developed a mass spectrometric sequencing strategy that allows the identification of in vivo O-glycosylation sites on mucin-derived glycopeptides. Lactation-associated MUC1 was isolated from human milk and partially deglycosylated by trifluoromethanesulfonic acid to the level of core GalNAc residues. The product was fragmented by the Arg-C-specific endopeptidase clostripain to yield tandem repeat icosapeptides starting with the PAP motif. PAP20 glycopeptides were subjected to sequencing by post-source decay matrix-assisted laser desorption ionization mass spectrometry or by solid phase Edman degradation to localize the glycosylation sites. The masses of C- or N-terminal fragments registered for the mono- to pentasubstituted PAP20 indicated that GalNAc was linked to the peptide at Ser5,Thr6 (GSTA) and Thr14(VTSA) but contrary to previous in vitro glycosylation studies also at Thr19 and Ser15 located within the PDTR or VTSA motifs, respectively. Quantitative data from solid phase Edman sequencing revealed no preferential glycosylation of the threonines. These discrepancies between in vivo andin vitro glycosylation patterns may be explained by assuming that O-glycosylation of adjacent peptide positions is a dynamically regulated process that depends on changes of the substrate qualities induced by glycosylation at vicinal sites.

The Importance of Protein Co- and Post-Translational Modifications in Proteome Projects

Any protein in a proteome can be modified by co- and post-translational modifications. There are literally dozens of different types of such modifications, all of which can influence a protein’s charge, hydrophobicity, conformation and/or stability. Hence, the “one-gene-one-polypeptide” paradigm is now outdated as we understand that in both eukaryotes and prokaryotes the polypeptide translation of many genes is modified to create multiple gene products from a single DNA sequence (Fig. 4.1). Well-documented examples include the differential glycosylation of Thy-1 expressed in different tissues (Parekh et al. 1987) and the differential RNA splicing of N-CAM (Cunningham et al. 1987).

Amino acid analysis protocols

Amino Acid Analysis: An Overview, Margaret I. Tyler. Amino Acid Analysis, Using Postcolumn Ninhydrin Detection, in a Biotechnology Laboratory, Frank D. Macchi, Felicity J. Shen, Rodney G. Keck, and Reed J. Harris. Purification of Proteins Using UltraMacro Spin Columns or ProSorb Sample Preparation Cartridges for Amino Acid Analysis, Li Zhang and Nancy Denslow. Amino Acid Analysis Using Precolumn Derivatization with 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate Steven A. Cohen. Amino Acid Analysis by High-Performance Liquid Chromatography after Derivatization with 1-Fluoro-2,4-Dinitrophenyl-5-l-Alanine Amide (Marfeys Reagent), Sunil Kochhar, Barbara Mouratou, and Philipp Christen. The Analysis of Amino Acids Using Precolumn Derivatization, HPLC, and Electrochemical Detection, C. David Forster and Charles A. Marsden. Anion Exchange Chromatography and Intergrated Amperometric Detection of Amino Acids, Petr Jandik, Christopher Pohl, Victor Barreto, and Nebojsa Avdalovic. Ion-Pair Chromatography for Identification of Picomolar-Order Protein on a PVDF Membrane, Noriko Shindo, Tsutomu Fujimura, Saiko Kazuno, and Kimie Murayama. Capillary Gas Chromatographic Analysis of Protein and Nonprotein Amino Acids in Biological Samples, Hiroyuki Kataoka, Sayuri Matsumura, Shigeo Yamamoto, and Masami Makita. Measurement of Blood Plasma Amino Acids in Ultrafiltrates by High-Performance Liquid Chromatography with Automatic Precolumn O-Phthaldialdehyde Derivatization, Hua Liu. Determination of Amino Acids in Foods by Reversed-Phase High-Performance Liquid Chromatography with New Precolumn Derivatives, Butylthiocarbamyl, and Benzylthiocarbamyl Derivatives Compared to the Phenylthiocarbamyl Derivative and Ion Exchange Chromatography, Kang-Lyung Woo. Amino Acid Measurement in Body Fluids Using PITC Derivatives, Roy A. Sherwood. Determination of Proteins, Phosphatidylethanolamine, and Phosphatidylserine in Lipid-Rich Materials by Analysis. of Phenylthiocarbamyl Derivatives, Margareta Stark and Jan Johansson. Analysis of O-Phosphoamino Acids in Biological Samples by Gas Chromatography with Flame Photometric Detection, Hiroyuki Kataoka, Norihisa Sakiyama, Yukizo Ueno, Kiyohiko Nakai, and Masami Makita. Determination of Sulfur Amino Acids, Glutathione, and Related Aminothiols in Biological Samples by Gas Chromatography with Flame Photometric Detection, Hiroyuki Kataoka, Kiyomi Takagi, Hirofumi Tanaka, and Masami Makita. Capillary Electrophoretic Determination of 4-Hydroxyproline, Qingyi Chu and Michael Zeece. Total Plasma Homocysteine Analysis by HPLC with SBD-F Precolumn Derivatization, Isabella Fermo and Rita Paroni. Determination of Early Glycation Products by Mass Spectrometry and Quantification of Glycation Mediated Protein Crosslinks by the Incorporation of [14C]lysine into Proteins, Malladi Prabhakaram, Beryl J. Ortwerth, and Jean B. Smith. Index.