Addressing the Protease Bias in Quantitative Proteomics.

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Label-free mass spectrometry often suffers from poor interprotease accuracy.
• Protease selection significantly impacts the reliability of protein quantification.

Purpose of the Study:

• To explore protease bias and its impact on accuracy and precision in protein quantification.
• To establish robust and reproducible methods for absolute quantification of human plasma proteins.

Main Methods:

• Utilized six different proteases with distinct cleavage sites.
• Employed recombinant protein standards and established 557 selected reaction monitoring (SRM) assays.
• Quantified 10 human plasma proteins across a concentration range of 0.02–70 μM.
• Used spiked stable isotope standard recombinant proteins in a targeted proteomics workflow.

Main Results:

• Demonstrated that utilizing multiple proteases can mitigate protease bias and enhance quantification accuracy.
• Achieved high robustness and reproducibility for the quantification of 60 peptides across 10 plasma proteins.
• Successfully quantified plasma proteins spanning three orders of magnitude.

Conclusions:

• Multiple proteases improve accuracy in label-free mass spectrometry for protein quantification.
• Targeted proteomics workflows with stable isotope standards offer robust and reproducible quantification of plasma proteins.