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).

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.

Figure 1

2D-Maps representing total protein extract from macrophages and protein fractions derived from the proposed protocol. 2D mini gels of a total protein extract from macrophages cells (panel on the left) and the 4 fractions obtained by applying the extraction protocol with Triton X114 (cytosolic fraction, membrane fraction, membrane associated fraction and secretome fractions, from the top to the bottom). All the 2D-PAGE are run on a 3–10 non linear IPG strip and an equal amount of total protein content was loaded on each gel. For each 2D map the spots count, as obtained with PDQuest software, is indicated.

Figure 2

Western blot analysis for PGK-1 presence in the obtained fractions. Western blot analyses for the cytosolic enzyme phosphoglycerate kinase (PGK I), typical of the glycolysis, panel A, and for the membrane bound protein Metalloproteinase 9 (MMP9), panel B. The three fractions were run together on the same SDS-PAGE loading an equal amount of total protein. The same PVDF membrane was used. Film image with the relative protein bands only in the lane corresponding to the cytosolic compartment and to the membrane fraction are reported. Western blot images were captured by GS710 densitometer (Bio-Rad) and analyzed by QuantityOne software.

Figure 3

Comparison between total extract 2D map and fractions 2D maps. 2D gels of total extract and fractions with enlargements of specific zones and a diagram reassuming the numerical data obtained. A) total extract versus membrane fraction. B) total extract versus cytosolic fraction. C) total extract versus membrane associated fraction. D) Pie chart representing the Spots counts relative to the total extract (left panel) and the summary of the 4 fractions obtained with the proposed protocol.

Figure 4

2D map of the membrane fraction used for spot excision and ID. 2D gel of the membrane fraction showing the cut spots as red circles numbered from 1 to 21.