Measuring ¹⁵N and ¹³C Enrichment Levels in Sparsely Labeled Proteins Using High-Resolution and Tandem Mass...

Area of Science:

• Biochemistry
• Structural Biology
• Analytical Chemistry

Background:

• Sparse isotope labeling (¹⁵N, ¹³C) is crucial for complex proteins like glycoproteins expressed in mammalian cells.
• High enrichment levels are essential for multidimensional heteronuclear NMR studies of protein structure.

Purpose of the Study:

• To develop and validate a mass spectrometry-based method for quantifying isotope label incorporation in sparsely labeled glycoproteins.
• To identify and overcome challenges in label incorporation determination, such as metabolic scrambling and medium dilution.

Main Methods:

• Utilized mass spectrometry to analyze tryptic peptides of selectively labeled glycoproteins.
• Developed a data analysis approach involving simulation of isotope patterns and linear combination fitting.
• Employed ultrahigh mass resolution and tandem mass spectrometry for precise enrichment level assignment and label localization.

Main Results:

• Identified complex isotope patterns due to metabolic scrambling, complicating direct analysis.
• Successfully quantified ¹⁵N and ¹³C label incorporation using the developed simulation and fitting method.
• Detected previously unrecognized metabolic scrambling effects impacting NMR experiments.

Conclusions:

• The developed mass spectrometry method accurately quantifies sparse isotope labeling in glycoproteins, even with metabolic scrambling.
• This approach enhances the reliability of NMR-based structural analysis of glycoproteins.
• The findings provide critical insights into metabolic pathways affecting isotope labeling in cell expression systems.