期刊
JOURNAL OF CHROMATOGRAPHY A
卷 1217, 期 19, 页码 3307-3315出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2009.09.029
关键词
High-performance multi-lectin affinity chromatography; Lectin blotting; Isoelectric focusing; Lectin-antibody microarray
资金
- NCI NIH HHS [U01 CA128427-03, R33 CA122890, U01 CA128427, U01-CA128427, U01 CA152653] Funding Source: Medline
Protein glycosylation represents one of the major post-translational modifications and can have significant effects on protein function. Moreover, changes in the carbohydrate structure are increasingly being recognized as an important modification associated with cancer etiology. In this report, we describe the development of a proteomics approach to identify breast cancer related changes in either concentration and/or the carbohydrate structures of glycoprotein(s) present in blood samples. Diseased and healthy serum samples were processed by an optimized sample preparation protocol using multiple lectin affinity chromatography (M-LAC) that partitions serum proteins based on glycan characteristics. Subsequently, three separate procedures, 1D SDS-PAGE, isoelectric focusing and an antibody microarray, were applied to identify potential candidate markers for future study. The combination of these three platforms is illustrated in this report with the analysis of control and cancer glycoproteomic fractions. Firstly, a molecular weight based separation of glycoproteins by 1D SDS-PAGE was performed, followed by protein, glycoprotein staining, lectin blotting and LC-MS analysis. To refine or confirm the list of interesting glycoproteins, isoelectric focusing (targeting sialic acid changes) and an antibody microarray (used to detect neutral glycan shifts) were selected as the orthogonal methods. As a result, several glycoproteins including alpha-1B-glycoprotein, complement 0, alpha-l-antitrypsin and transferrin were identified as potential candidates for further study. (C) 2009 Elsevier B.V All rights reserved.
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