Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myelo\"id cell lines nuclear proteomes

TL;DR

This study enhances nuclear proteomic analysis by optimizing extraction methods and applying 2D-gel electrophoresis to compare nuclear proteins in macrophages and dendritic cells, revealing modifications and low-abundance proteins.

Contribution

It introduces an improved protocol for nuclear protein extraction and demonstrates its effectiveness in analyzing nuclear proteomes with 2D-gel electrophoresis, including post-translational modifications.

Findings

Successful visualization of low-abundance nuclear proteins

Identification of post-translational modifications affecting protein profiles

Detection of chromatin-related proteins like HP1 in nuclear extracts

Abstract

One of the challenges of the proteomic analysis by 2D-gel is to visualize the low abundance proteins, particularly those localized in organelles. An additional problem with nuclear proteins lies in their strong interaction with nuclear acids. Several experimental procedures have been tested to increase, in the nuclear extract, the ratio of nuclear proteins compared to contaminant proteins, and also to obtain reproducible conditions compatible with 2D-gel electrophoresis. The NaCl procedure has been chosen. To test the interest of this procedure, the nuclear protein expression profiles of macrophages and dendritic cells have been compared with a proteomic approach by 2D-gel electrophoresis. Delta 2D software and mass spectrometry analyses have allowed pointing out some proteins of interest. We have chosen some of them, involved in transcriptional regulation and/or chromatin structure for further validations. The immunoblotting experiments have shown that most of observed changes are due to post-translational modifications, thereby a exemplifying the interest of the 2D gel approach. Finally, this approach allowed us to reach not only high abundance nuclear proteins but also lower abundance proteins, such as the HP1 proteins and reinforces the interest of using 2DE-gel in proteomics because of its ability to visualize intact proteins with their modifications.