title: High resolution preparation of monocyte-derived macrophages (MDM) protein fractions for clinical proteomics.
abstract: BACKGROUND
  Macrophages are involved in a number of key physiological processes and complex responses such as inflammatory, immunological, infectious diseases and iron homeostasis. These cells are specialised for iron storage and recycling from senescent erythrocytes so they play a central role in the fine tuning of iron balancing and distribution. The comprehension of the many physiological responses of macrophages implies the study of the related molecular events. To this regard, proteomic analysis, is one of the most powerful tools for the elucidation of the molecular mechanisms, in terms of changes in protein expression levels.
  
  
  RESULTS
  Our aim was to optimize a protocol for protein fractionation and high resolution mapping using human macrophages for clinical studies. We exploited a fractionation protocol based on the neutral detergent Triton X-114. The 2D maps of the fractions obtained showed high resolution and a good level of purity. Western immunoblotting and mass spectrometry (MS/MS analysis) indicated no fraction cross contamination. On 2D-PAGE mini gels (7 x 8 cm) we could count more than five hundred protein spots, substantially increasing the resolution and the number of detectable proteins for the macrophage proteome. The fractions were also evaluated, with preliminary experiments, using Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS).
  
  
  CONCLUSIONS
  This relatively simple method allows deep investigation into macrophages proteomics producing discrete and accurate protein fractions, especially membrane-associated and integral proteins. The adapted protocol seems highly suitable for further studies of clinical proteomics, especially for the elucidation of the molecular mechanisms controlling iron homeostasis in normal and disease conditions.
authors.affiliation: University of Verona, Department of Biotechnology, Italy. rita.polati@univr.it
authors.forename: Rita
authors.initials: R
authors.lastname: Polati
authors.forename: Annalisa
authors.initials: A
authors.lastname: Castagna
authors.forename: Alessandra
authors.initials: A
authors.lastname: Bossi
authors.forename: Natascia
authors.initials: N
authors.lastname: Campostrini
authors.forename: Federica
authors.initials: F
authors.lastname: Zaninotto
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Timperio
authors.forename: Lello
authors.initials: L
authors.lastname: Zolla
authors.forename: Oliviero
authors.initials: O
authors.lastname: Olivieri
authors.forename: Roberto
authors.initials: R
authors.lastname: Corrocher
authors.forename: Domenico
authors.initials: D
authors.lastname: Girelli