SILAC-Based Quantitative Strategies for Accurate Histone Posttranslational Modification Profiling Across Multiple...

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Histone posttranslational modifications (hPTMs) are crucial for chromatin regulation and DNA processes.
• Mass spectrometry (MS) is a powerful tool for analyzing histones and identifying hPTMs.
• Stable isotope labeling by amino acid in cell culture (SILAC) enables quantitative analysis of PTMs but is limited to dividing cells.

Purpose of the Study:

• To adapt SILAC for comprehensive histone analysis, including non-dividing cells.
• To develop a spike-in SILAC strategy for profiling hPTMs across multiple samples.
• To integrate SILAC with a shotgun proteomics workflow for detailed hPTM analysis.

Main Methods:

• Adaptation of SILAC for histone analysis in standard and spike-in configurations.
• Implementation of a shotgun proteomics workflow involving Arg-C digestion.
• Qualitative and quantitative analysis of labeled histone peptides using LC-MS/MS with UHPLC and Orbitrap high-resolution mass spectrometry.

Main Results:

• Successful adaptation of SILAC for histone analysis, overcoming limitations of cell division.
• Demonstration of a spike-in SILAC strategy for robust hPTM profiling across multiple samples.
• Effective coupling of SILAC with a shotgun workflow for in-depth peptide analysis.

Conclusions:

• The adapted SILAC method provides a versatile approach for quantitative hPTM analysis.
• The spike-in SILAC strategy broadens the applicability of SILAC to various cell types and experimental designs.
• This integrated workflow enhances the ability to study histone modifications and their functional implications.