Publications

Authors:

Polati, Rita; Castagna, Annalisa; Bossi, Alessandra Maria; Campostrini, Natascia; Zaninotto, Federica; Timperio, A. M.; Zolla, L.; Olivieri, Oliviero; Corrocher, Roberto; Girelli, Domenico

Title:

High resolution preparation of monocyte-derived macrophages (MDM) protein fractions for clinical proteomics

Year:

2009

Type of item:

Articolo in Rivista

Tipologia ANVUR:

Articolo su rivista

Language:

Inglese

Format:

Elettronico

Referee:

Sì

Name of journal:

PROTEOME SCIENCE

ISSN of journal:

1477-5956

N° Volume:

7

Page numbers:

1-11

Keyword:

MASS-SPECTROMETRY; MEMBRANE-PROTEINS; HEPCIDIN; ELECTROPHORESIS; FERROPORTIN; EXTRACTION; DISEASE; BINDING

Short description of contents:

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 × 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). Conclusion: 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.

Product ID:

48931

Handle IRIS:

11562/326440

Deposited On:

July 4, 2012

Last Modified:

November 30, 2022

Bibliographic citation:

Polati, Rita; Castagna, Annalisa; Bossi, Alessandra Maria; Campostrini, Natascia; Zaninotto, Federica; Timperio, A. M.; Zolla, L.; Olivieri, Oliviero; Corrocher, Roberto; Girelli, Domenico, High resolution preparation of monocyte-derived macrophages (MDM) protein fractions for clinical proteomics «PROTEOME SCIENCE» , vol. 7 , 2009 , pp. 1-11

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