title: Proteomic analysis to identify breast cancer biomarkers in nipple aspirate fluid.
abstract: OBJECTIVE
  Proteomic analysis of breast nipple aspirate fluid (NAF) holds promise as a noninvasive method to identify markers of breast cancer. The objectives of the study were to: (a) describe the NAF proteome, (b) identify candidate markers of breast cancer in NAF by using proteomic analysis, and (c) validate the markers identified by using a quantitative, high-throughput ELISA analysis.
  
  
  METHODS
  For proteome analysis, NAF proteins from a single subject without breast cancer were separated by two-dimensional PAGE and were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectometry identification. A total of 41 different proteins were identified, 25 of which were known to be secreted. To identify breast cancer markers, we separated 20 NAF samples (10 normal, 10 cancer) by two-dimensional PAGE. Three protein spots were detected that were up-regulated in three or more cancer samples. These spots were identified to be gross cystic disease fluid protein (GCDFP)-15, apolipoprotein D (apoD), and alpha1-acid glycoprotein (AAG). To validate these three potential biomarkers, 105 samples (53 from benign breasts and 52 from breasts with cancer) were analyzed using ELISA.
  
  
  RESULTS
  Among all of the subjects, GCDFP-15 levels were lower (P <0.001) and AAG levels were higher (P=0.001) in breasts with cancer. This was also true in premenopausal (GCDFP-15, P=0.011; AAG, P=0.002) but not in postmenopausal women. GCDFP-15 levels were lowest (P=0.003) and AAG levels highest (P <0.001) in women with ductal carcinoma in situ (DCIS). Menopausal status influenced GCDFP-15 and AAG more in women without breast cancer than in women with breast cancer. apoD levels did not correlate significantly with breast cancer.
  
  
  CONCLUSIONS
  Our study revealed that the NAF proteome, as defined by two-dimensional PAGE, consists of a limited number of proteins, and that the expression of AAG and GCDFP-15 correlates with disease presence and stage.
authors.affiliation: Division of Biological Sciences, and Department of Surgery, University of Missouri, Columbia, Missouri 65212, USA.
authors.forename: Hannah
authors.initials: H
authors.lastname: Alexander
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Stegner
authors.forename: Colette
authors.initials: C
authors.lastname: Wagner-Mann
authors.forename: Garrett C
authors.initials: GC
authors.lastname: Du Bois
authors.forename: Stephen
authors.initials: S
authors.lastname: Alexander
authors.forename: Edward R
authors.initials: ER
authors.lastname: Sauter
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001053:Apolipoproteins / Q000096:biosynthesis; D053399:Apolipoproteins D; D014408:Biomarkers, Tumor / Q000378:metabolism*; D001707:Biopsy, Needle; D001826:Body Fluids / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism*; D002352:Carrier Proteins / Q000096:biosynthesis; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006023:Glycoproteins / Q000096:biosynthesis; D006801:Humans; D026901:Membrane Transport Proteins; D008593:Menopause; D008875:Middle Aged; D009961:Orosomucoid / Q000096:biosynthesis; D011379:Prognosis; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015854:Up-Regulation
chemical_list: D001053:Apolipoproteins; D053399:Apolipoproteins D; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D006023:Glycoproteins; D026901:Membrane Transport Proteins; D009961:Orosomucoid; C488892:PIP protein, human