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Biomarker discovery in breast cancer serum using 2‐D differential gel electrophoresis/ MALDI‐TOF/TOF and data validation by routine clinical assays
Author(s) -
Huang HongLei,
Stasyk Taras,
Morandell Sandra,
Dieplinger Hans,
Falkensammer Gerda,
Griesmacher Andrea,
Mogg Maurice,
Schreiber Martin,
Feuerstein Isabel,
Huck Christian W.,
Stecher Guenther,
Bonn Guenther K.,
Huber Lukas A.
Publication year - 2006
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200500857
Subject(s) - haptoglobin , transferrin , biomarker , apolipoprotein b , biomarker discovery , chemistry , difference gel electrophoresis , ceruloplasmin , proteomics , blood proteins , gel electrophoresis , albumin , apolipoprotein a1 , proteome , microbiology and biotechnology , biology , biochemistry , immunology , cholesterol , gene
In the present study, we used 2‐D differential gel electrophoresis (2‐D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39 patients with breast cancer and 35 controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low‐abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2‐D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A‐I, transferrin, and hemoglobin as up‐regulated and three spots, apolipoprotein A‐I, apolipoprotein C‐III, and haptoglobin α2 as down‐regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2‐D‐DIGE results. However, the serum levels of apolipoprotein A‐I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2‐D DIGE still has over other techniques. 2‐D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform‐special antibodies.