Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myeloid cell lines nuclear proteomes

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.3

TSR2 Induces laryngeal cancer cell apoptosis through inhibiting NF‐κB signaling pathway

“…Nuclear and cytosolic proteins were extracted with the Nuclear/Cytosol Fractionation Kit (BioVision, San Francisco, CA) according to the manufacturer's protocol . Briefly, cells were washed with phosphate‐buffered saline twice, followed by centrifuging at 4 °C (600 g ) for 5 minutes.…”

“…Nuclear and cytosolic proteins were extracted with the Nuclear/Cytosol Fractionation Kit (BioVision, San Francisco, CA) according to the manufacturer's protocol. 8 Briefly, cells were washed with phosphate-buffered saline twice, followed by centrifuging at 4 8C (600g) for 5 minutes. The supernatant was thrown away, and the precipitate was lightly suspended with cytosolic extraction buffer on ice for 15 minutes followed by centrifuging at 4 8C (14,000g) for 15 minutes.…”

Transcription factor proteomics—Tools, applications, and challenges

“…The method that will ultimately be selected depends largely on the scope of the study. Nevertheless, it is important to emphasize that it remains challenging to efficiently enrich or extract chromatin‐associated proteins under conditions that are compatible with mass spectrometry, independent of the chosen assay . One important consequence is the elevated risk for underestimating protein amounts when dealing with TFs.…”

Preparing Protein Extracts for Quantitative Two‐Dimensional Gel Comparison

“…Given so many constraints, a complete and universal protein solubilization method for two-dimensional electrophoresis is an almost unreachable Holy Grail. For example, separating proteins from nucleic acids and removing the latter without carrying over proteins usually requires high salt concentrations and/or ionic detergents or chaotropes, conditions incompatible with two-dimensional electrophoresis unless a sequential salt extraction followed by a precipitation step is performed (e.g., in Lelong et al, 2012). As another example, removal of lipids is usually achieved by extraction into organic solvents, but this precipitates proteins.…”