Quantifying In Situ Structural Stabilities of Human Blood Plasma Proteins Using a Novel Iodination Protein Stability...

Area of Science:

• Biochemistry and Molecular Biology
• Proteomics and Mass Spectrometry
• Structural Biology and Biophysics

Background:

• Protein quality loss is a significant driver of many diseases.
• Protein folding stability (PFS) is a key indicator of protein quality.
• Current methods for measuring PFS have limitations in protein coverage and granularity.

Purpose of the Study:

• To introduce a novel mass spectrometry-based method, the iodination protein stability assay (IPSA), for quantifying protein folding stability (PFS).
• To demonstrate IPSA's capability for measuring PFS in human serum proteins in situ.
• To present an analysis tool, C_half, for calculating residue-specific PFS.

Main Methods:

• Developed the iodination protein stability assay (IPSA), a mass spectrometry-based technique.
• IPSA quantifies PFS by tracking surface accessibility of specific amino acid residues (tyrosine, histidine, methionine, cysteine) under denaturing conditions.
• Utilized the C_half analysis tool for residue-specific PFS calculations.

Main Results:

• IPSA provides increased protein coverage and sequence granularity compared to existing methods.
• Demonstrated the first in situ measurement of PFS for human serum proteins.
• Quantified PFS differences in transferrin based on iron-binding states under near in vivo conditions, showing residue-specific PFS reporting.

Conclusions:

• IPSA offers a novel and improved approach for measuring protein folding stability.
• The method enables residue-specific PFS analysis and in situ measurements of serum proteins.
• IPSA has the potential to advance the use of protein structural stability as a metric for understanding disease etiology and progression.