Using stable-isotope-labeled proteins for hydrogen exchange studies in complex mixtures

Area of Science:

• Biochemistry
• Analytical Chemistry
• Structural Biology

Background:

• Studying protein dynamics in complex biological systems is challenging.
• Existing methods struggle to isolate and analyze individual proteins within heterogeneous mixtures.
• Understanding protein hydrogen exchange rates is crucial for elucidating protein structure and function.

Purpose of the Study:

• To develop a mass spectrometry-based method for measuring hydrogen exchange rates of specific proteins in complex mixtures.
• To utilize stable-isotope labeling (SIL) for unambiguous identification of target peptides.
• To demonstrate the applicability of this method in complex biological samples like bacterial cell lysates.

Main Methods:

• Incorporation of stable-isotope-labeled (SIL) amino acids into the protein of interest during overexpression in bacteria.
• Analysis of peptic peptides using mass spectrometry to identify distinctive SIL isotope patterns.
• Measurement of hydrogen exchange rates for SIL-labeled peptides within complex protein mixtures.

Main Results:

• Distinctive isotope patterns in mass spectra served as a unique signature for peptides from the SIL-labeled protein.
• Stable-isotope labeling simplified peptide identification and provided partial amino acid composition.
• Hydrogen exchange rates for SIL-labeled peptides were successfully measured and found to be consistent with unlabeled proteins.
• Individual protein hydrogen exchange was accurately determined within a complex bacterial cell lysate.

Conclusions:

• This novel methodology allows for the facile recognition and analysis of specific peptides within complex proteomes.
• It enables the measurement of hydrogen exchange rates for individual proteins in heterogeneous systems.
• The technique is valuable for analyzing the structural properties and dynamics of large protein complexes and intricate biological systems.