title: Proteomic profiling of nipple aspirate fluid (NAF): Exploring the complementarity of different peptide fractionation strategies.
abstract: NAF is a breast fluid that is closely related to the tumor microenvironment and a valuable sample for studying breast cancer. To perform an in-depth proteomic analysis of this sample, aliquots of a single NAF digest were analyzed by the following peptide-centric fractionation strategies: a) 30-cm reversed-phase (RP) column on-line with an LTQ-Orbitrap XL; b) off-line strong cation-exchange (SCX) column; and c) pI-based OFFGEL fractionation. All fractions from approaches (b) and (c) were further analyzed on a 10-cm RP column hyphenated to the same mass spectrometer. The RP-30cm, SCX/RP-10cm, and OFFGEL/RP-10cm approaches identified 1676, 2930, and 3240 peptides, which corresponded to 193, 390 and 528 proteins, respectively. In our cumulative dataset, 4466 distinct NAF peptides corresponded to a total of 557 proteins, of which only 34% were identified by all three approaches. No exclusive protein identification was associated to the RP-30cm approach, while SCX/RP-10cm and OFFGEL/RP-10cm contributed to 28 and 166 exclusive identifications, respectively. Each approach provided additional information related to energy metabolism (fermentation process/carbohydrate biosynthesis). In conclusion, the pre-fractionation platforms used were complementary for the comprehensive characterization of NAF and our work provides methodological information for future quantitative cancer-related NAF sample studies.
  
  
  CONCLUSIONS
  High-resolution peptide separation is a sine qua non condition for achieving extensive proteome coverage. Various techniques have been employed to improve peptide fractionation prior to LC-MS/MS, thus allowing a comprehensive characterization of complex biological samples. Although fractionation efficiency is very sample-dependent, this issue is commonly overlooked, and a "cookbook" approach is routinely used during this critical step. The present study provides a systematic comparison of analytical information needed for the successful large-scale differential proteomic analysis of NAF samples from breast cancer patients, a precious and volume-limited biological sample. It reinforces the importance of optimizing sample-specific fractionation protocols for information retrieval from mass spectrometric analysis.
authors: Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
authors: Giselle Villa Flor
authors: GV
authors: Brunoro
authors: Laboratory for Proteomics and Protein Engineering, Carlos Chagas Institute, Fiocruz, Paraná, Brazil.
authors: Paulo Costa
authors: PC
authors: Carvalho
authors: Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
authors: André Teixeira da Silva
authors: AT
authors: Ferreira
authors: Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
authors: Jonas
authors: J
authors: Perales
authors: Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
authors: Richard Hemmi
authors: RH
authors: Valente
authors: Laboratory of Molecular Biology of Tumors, Department of Genetics, State University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors: Claudia Vitória
authors: CV
authors: de Moura Gallo
authors: Laboratory of Applied Molecular Biology, Gynecology Department, Fernandes Figueira Institute, Fiocruz, Rio de Janeiro, Brazil.
authors: Dante
authors: D
authors: Pagnoncelli
authors: Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil. Electronic address: anag@ioc.fiocruz.br.
authors: Ana Gisele da Costa
authors: AG
authors: Neves-Ferreira
mesh_terms: D000328:Adult; D005260:Female; D018226:Fibroadenoma / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D009363:Neoplasm Proteins / Q000378:metabolism*; D055966:Nipple Aspirate Fluid / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D040901:Proteomics; D059016:Tumor Microenvironment
chemical_list: D009363:Neoplasm Proteins; D010455:Peptides
keywords: Breast fluid; NAF; Pre-fractionation; Shotgun