TY - JOUR
T1 - The development of an integrated platform to identify breast cancer glycoproteome changes in human serum
AU - Zeng, Zhi
AU - Hincapie, Marina
AU - Haab, Brian B.
AU - Hanash, Samir
AU - Pitteri, Sharon J.
AU - Kluck, Steven
AU - Hogan, Jason M.
AU - Kennedy, Jacob
AU - Hancock, William S.
N1 - Funding Information:
This study was supported by National Cancer Institute grant U01-CA128427 . The authors thank Russell Garlick, Jim Dasch, Oren Kagan and Andrew Johnson from Protein Forest, Inc. for providing d PC, technical support and helpful discussion. We are grateful to Dr. Shiaw-Lin Wu and Dr. Tomas Rejtar for advice on this project. Thanks also to Majlinda Kullolli and Manohar Akella for technical support.
PY - 2010/5
Y1 - 2010/5
N2 - 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 C3, alpha-1-antitrypsin and transferrin were identified as potential candidates for further study.
AB - 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 C3, alpha-1-antitrypsin and transferrin were identified as potential candidates for further study.
KW - High-performance multi-lectin affinity chromatography
KW - Isoelectric focusing
KW - Lectin blotting
KW - Lectin-antibody microarray
UR - http://www.scopus.com/inward/record.url?scp=77951948977&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77951948977&partnerID=8YFLogxK
U2 - 10.1016/j.chroma.2009.09.029
DO - 10.1016/j.chroma.2009.09.029
M3 - Article
C2 - 19782370
AN - SCOPUS:77951948977
SN - 0021-9673
VL - 1217
SP - 3307
EP - 3315
JO - Journal of Chromatography A
JF - Journal of Chromatography A
IS - 19
ER -