Comparative proteomics of glycoproteins based on lectin selection and isotope coding.
Lectins have been widely used in glycan structure analysis. The studies described here exploit this fact to select glycopeptides carrying disease-associated modifications in their oligosaccharides. Coupling lectin affinity selection with recent advances in stable isotope coding for quantitative proteomics allowed a comparative proteomics method to be developed for examining aberrant glycosylation in cancer. Control and experimental samples were individually tryptic digested and differentially coded with stable isotope coding agents before they were mixed and affinity selected with a lectin affinity chromatography column. Glycopeptides carrying an alpha-L-fucose residue were selected with Lotus tetragonolobus agglutinin (LTA) immobilized on a chromatography matrix. Because the oligosaccharides of glycoproteins are generally heterogeneous and often of unknown structure, it was necessary to deglycosylate the selected peptides with PNGase F before they could be compared to sequences in DNA and protein databases. After deglycosylated peptides were transferred to a reversed phase chromatography (RPC) column and fractionated by gradient elution with increasing amounts of acetonitrile. The RPC fractions were then analyzed by both matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS). When this method was applied to a study of lymphosarcoma in canines, it was found that during chemotherapy, a series of fucosylated proteins in the blood of patients decreased in concentration more than 2-fold. Two of the proteins identified, CD44 and E-selectin, are known to be involved in cell adhesion and cancer cell migration. The observed aberrant fucosylation of these proteins is consistent with the hypothesis that CD44 and E-selectin play a key role in metastasis and the spread of cancer cells to remote sites.
Xiong
Li
L
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Andrews
Dina
D
Regnier
Fred
F
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D004285:Dogs; D004358:Drug Therapy; D019040:E-Selectin / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000378:metabolism; D007554:Isotopes / Q000737:chemistry*; D037102:Lectins / Q000378:metabolism*; D008228:Lymphoma, Non-Hodgkin / Q000378:metabolism; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D019040:E-Selectin; D006023:Glycoproteins; D018960:Hyaluronan Receptors; D007554:Isotopes; D037102:Lectins; D020033:Protein Isoforms