Two-dimensional gel proteome reference map of blood monocytes

Abstract

Background: Blood monocytes play a central role in regulating host inflammatory processes through chemotaxis, phagocytosis, and cytokine production. However, the molecular details underlying these diverse functions are not completely understood. Understanding the proteomes of blood monocytes will provide new insights into their biological role in health and diseases.

Results: In this study, monocytes were isolated from five healthy donors. Whole monocyte lysates from each donor were then analyzed by 2D gel electrophoresis, and proteins were detected using Sypro Ruby fluorescence and then examined for phosphoproteomes using ProQ phospho-protein fluorescence dye. Between 1525 and 1769 protein spots on each 2D gel were matched, analyzed, and quantified. Abundant protein spots were then subjected to analysis by mass spectrometry. This report describes the protein identities of 231 monocyte protein spots, which represent 164 distinct proteins and their respective isoforms or subunits. Some of these proteins had not been previously characterized at the protein level in monocytes. Among the 231 protein spots, 19 proteins revealed distinct modification by protein phosphorylation.

Conclusion: The results of this study offer the most detailed monocyte proteomic database to date and provide new perspectives into the study of monocyte biology.

Figure 1

Demonstration of monocyte 2D gel reproducibility between runs and visual examination of proteome analysis of all five 2D gels using the montage window of a specific gel area in blood monocytes. (A) Monocyte 2D gel from donor 1. (B) Magnified image of the same gel area for all five monocyte 2D gels.

Figure 2

Monocyte proteome map. (A) 2D gel of blood monocytes. Arrows point to abundant proteins with protein identity determined by tandem mass spectrometry. (B) Detailed illustration of the central area of Figure 2A. Arrows illustrate protein spots in the central area that have information on protein identity.

Figure 3

Analysis of monocyte phosphoproteome by ProQ staining. (A) Specificity of pro Q stain for phosphorylated proteins. Lane 1: Pro Q staining of monocyte lysates. Lane 2: Same cell lysates treated with protein phosphatase (80 units/ml) for 60 minutes at 30°C prior to SDS-PAGE/Pro Q staining. Lane 3: Sypro Ruby fluorescence staining of Lane 1. Lane 4: Sypro Ruby fluorescence staining of Lane 2. A protein band at about 25 kDa is consistent with M_r of protein phosphatase added to the sample. (B) A 2D gel of monocytes stained with Pro Q diamond phosphoprotein dye. Arrows point to the protein spots with IDs and positive for phosphorylation stain.