title: High-throughput proteomic analysis of human infiltrating ductal carcinoma of the breast.
abstract: Large-scale proteomics will play a critical role in the rapid display, identification and validation of new protein targets, and elucidation of the underlying molecular events that are associated with disease development, progression and severity. However, because the proteome of most organisms are significantly more complex than the genome, the comprehensive analysis of protein expression changes will require an analytical effort beyond the capacity of standard laboratory equipment. We describe the first high-throughput proteomic analysis of human breast infiltrating ductal carcinoma (IDCA) using OCT (optimal cutting temperature) embedded biopsies, two-dimensional difference gel electrophoresis (2-D DIGE) technology and a fully automated spot handling workstation. Total proteins from four breast IDCAs (Stage I, IIA, IIB and IIIA) were individually compared to protein from non-neoplastic tissue obtained from a female donor with no personal or family history of breast cancer. We detected differences in protein abundance that ranged from 14.8% in stage I IDCA versus normal, to 30.6% in stage IIB IDCA versus normal. A total of 524 proteins that showed > or = three-fold difference in abundance between IDCA and normal tissue were picked, processed and identified by mass spectrometry. Out of the proteins picked, approximately 80% were unambiguously assigned identities by matrix-assisted laser desorbtion/ionization-time of flight mass spectrometry or liquid chromatography-tandem mass spectrometry in the first pass. Bioinformatics tools were also used to mine databases to determine if the identified proteins are involved in important pathways and/or interact with other proteins. Gelsolin, vinculin, lumican, alpha-1-antitrypsin, heat shock protein-60, cytokeratin-18, transferrin, enolase-1 and beta-actin, showed differential abundance between IDCA and normal tissue, but the trend was not consistent in all samples. Out of the proteins with database hits, only heat shock protein-70 (more abundant) and peroxiredoxin-2 (less abundant) displayed the same trend in all the IDCAs examined. This preliminary study demonstrates quantitative and qualitative differences in protein abundance between breast IDCAs and reveals 2-D DIGE portraits that may be a reflection of the histological and pathological status of breast IDCA.
authors: Clinical Breast Care Project, Windber Research Institute, Windber PA 15963, USA. r.somiari@wriwindber.org
authors: Richard I
authors: RI
authors: Somiari
authors: Anthony
authors: A
authors: Sullivan
authors: Stephen
authors: S
authors: Russell
authors: Stella
authors: S
authors: Somiari
authors: Hai
authors: H
authors: Hu
authors: Rick
authors: R
authors: Jordan
authors: Alisha
authors: A
authors: George
authors: Richard
authors: R
authors: Katenhusen
authors: Alicja
authors: A
authors: Buchowiecka
authors: Cletus
authors: C
authors: Arciero
authors: Henry
authors: H
authors: Brzeski
authors: Jeff
authors: J
authors: Hooke
authors: Craig
authors: C
authors: Shriver
mesh_terms: D000328:Adult; D000368:Aged; D001053:Apolipoproteins / Q000032:analysis; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000378:metabolism* / Q000473:pathology; D019295:Computational Biology; D030562:Databases, Protein; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis; D006801:Humans; D007526:Isoelectric Point; D008875:Middle Aged; D008970:Molecular Weight; D009367:Neoplasm Staging; D010544:Peroxidases / Q000032:analysis; D054464:Peroxiredoxins; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015854:Up-Regulation; D000515:alpha 1-Antitrypsin / Q000032:analysis
chemical_list: D001053:Apolipoproteins; D018840:HSP70 Heat-Shock Proteins; D020543:Proteome; D000515:alpha 1-Antitrypsin; D010544:Peroxidases; D054464:Peroxiredoxins