The development of an integrated platform to identify breast cancer glycoproteome changes in human serum |
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Authors: | Zhi Zeng Marina Hincapie Brian B. Haab Samir Hanash Sharon J. Pitteri Steven Kluck Jason M. Hogan Jacob Kennedy William S. Hancock |
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Affiliation: | 1. Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA;2. Van Andel Research Institute, 333 Bostwick, Grand Rapids, MI 49503, USA;3. Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA |
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Abstract: | 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. |
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Keywords: | A1BG, alpha-1B-glycoprotein A1AT, alpha-1-antitrypsin AAL, Aleuria aurantia lectin ANT3, antithrombin-III BC, breast cancer CO3, complement C3 CO4A, complement C4-A CPAS, Computational Proteomics Analysis System dPC, digital ProteomeChip FA, formic acid HEMO, hemopexin HP-MLAC, high-performance multi-lectin affinity chromatography IAA, iodoacetamide IEF, isoelectric focusing MARS, multiple affinity removal system MeCN, acetonitrile MS, mass spectrometry MSRAT, Mass Spec Results Analysis Tool PBST-0.1, PBS buffer containing 0.1% Tween-20 PBST-0.5, PBS buffer containing 0.5% Tween-20 pI, isoelectric point SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis SNA, Sambucus Nigra Lectin TBST, tris-buffered saline Tween 20 TFA, trifluoroacetic acid TRFE, transferrin Xcorr, correlation score |
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