Comparative serum glycoproteomics using lectin selected sialic acid glycoproteins with mass spectrometric analysis: application to pancreatic cancer serum.
A strategy is developed in this study for identifying sialylated glycoprotein markers in human cancer serum. This method consists of three steps: lectin affinity selection, a liquid separation and characterization of the glycoprotein markers using mass spectrometry. In this work, we use three different lectins (Wheat Germ Agglutinin, (WGA) Elderberry lectin,(SNA), Maackia amurensis lectin, (MAL)) to extract sialylated glycoproteins from normal and cancer serum. Twelve highly abundant proteins are depleted from the serum using an IgY-12 antibody column. The use of the different lectin columns allows one to monitor the distribution of alpha(2,3) and alpha(2,6) linkage type sialylation in cancer serum vs that in normal samples. Extracted glycoproteins are fractionated using NPS-RP-HPLC followed by SDS-PAGE. Target glycoproteins are characterized further using mass spectrometry to elucidate the carbohydrate structure and glycosylation site. We applied this approach to the analysis of sialylated glycoproteins in pancreatic cancer serum. Approximately 130 sialylated glycoproteins are identified using microLC-MS/MS. Sialylated plasma protease C1 inhibitor is identified to be down-regulated in cancer serum. Changes in glycosylation sites in cancer serum are also observed by glycopeptide mapping using microLC-ESI-TOF-MS where the N83 glycosylation of alpha1-antitrypsin is down regulated. In addition, the glycan structures of the altered proteins are assigned using MALDI-QIT-MS. This strategy offers the ability to quantitatively analyze changes in glycoprotein abundance and detect the extent of glycosylation alteration as well as the carbohydrate structure that correlate with cancer.
Zhao
Jia
J
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Simeone
Diane M
DM
Heidt
David
D
Anderson
Michelle A
MA
Lubman
David M
DM
D016022:Case-Control Studies; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D037102:Lectins / Q000737:chemistry*; D013058:Mass Spectrometry; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D010190:Pancreatic Neoplasms / Q000737:chemistry*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods
D006023:Glycoproteins; D037102:Lectins; D020543:Proteome; D019158:N-Acetylneuraminic Acid