Detailed Method for Performing the ExSTA Approach in Quantitative Bottom-Up Plasma Proteomics

Area of Science:

• Proteomics
• Analytical Chemistry
• Biomarker Discovery

Background:

• Stable isotope-labeled standards (SIS) are crucial for accurate protein quantification in biological matrices.
• Various proteomic workflows and quantification strategies exist, with external standard addition (ExSTA) showing promise for limited sample volumes.

Purpose of the Study:

• To provide a detailed protocol for quantifying a multiplexed panel of 125 high-to-moderate abundance proteins in human plasma using the ExSTA method.
• To enable rapid and precise assessment of biomarker efficacy for proteins associated with noncommunicable diseases.

Main Methods:

• External standard addition (ExSTA) utilizing multipoint calibration curves generated with natural (NAT) peptides and fixed concentrations of SIS peptides.
• Spiking equal concentrations of SIS peptides into experimental sample digests.
• Solid-phase extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
• Determination of endogenous peptide concentrations via regression analysis of standard curves.

Main Results:

• A comprehensive protocol for ExSTA-based quantification of 125 proteins in undepleted, non-enriched human plasma is presented.
• The method allows for accurate protein quantification using calibration curves derived from external standards.
• Procedural details and recommendations for successful execution of the ExSTA workflow are described.

Conclusions:

• External standard addition (ExSTA) is an analytically valid and efficient strategy for large-scale protein quantification in biological samples.
• This detailed protocol facilitates the application of ExSTA for assessing biomarker efficacy in human plasma, particularly for noncommunicable disease research.
• The ExSTA method supports precise and rapid quantification crucial for biomarker validation in large cohort studies.