Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• Precise quantification of endogenous plasma proteins is crucial for research and clinical applications.
• Mass spectrometry-based targeted peptide detection with stable isotope-labeled peptides is a common quantitation approach.
• Measuring plasma proteins is challenging due to unknown endogenous levels, hindering optimal calibration strategies.

Purpose of the Study:

• To compare existing calibration strategies for endogenous plasma protein quantification.
• To introduce and evaluate a novel calibration approach using dual stable isotope-labeled standard (SIS) peptide isotopologues.
• To enhance the accuracy and robustness of plasma protein assays.

Main Methods:

• Comparison of various calibration strategies for endogenous plasma protein measurement.
• Development of a new method utilizing two distinct SIS peptide isotopologues per endogenous peptide.
• Preparation of external calibration curves and quality control samples in pooled human plasma without endogenous interference.

Main Results:

• The novel dual SIS peptide approach enables external calibration curve generation.
• Quality control samples can be prepared in pooled human plasma without endogenous interference.
• This strategy significantly improves analytical performance and assay accuracy monitoring.

Conclusions:

• The dual SIS peptide isotopologue strategy offers a superior method for endogenous plasma protein quantification.
• This approach enhances assay accuracy, facilitates method development, and aids in validation.
• It addresses the limitations of existing calibration strategies in plasma proteomics.