High-throughput absolute quantification of proteins using an improved two-dimensional reversed-phase separation and...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Stable isotope dilution-selective reaction monitoring-mass spectrometry (SID-SRM-MS) is a key technique for absolute protein quantification.
• Analyzing complex samples like mouse liver requires high-throughput methods for internal standard preparation and advanced separation techniques.

Purpose of the Study:

• To develop a high-throughput absolute protein quantification strategy for complex biological samples.
• To improve the efficiency and coverage of mass spectrometry-based proteomic analyses.

Main Methods:

• Utilized an improved two-dimensional reversed-phase (2D RP) separation method.
• Employed a quantification concatemer (QconCAT) approach with synthesized peptides from glutathione S-transferase (GST).
• Integrated QconCAT standards with digested complex samples for simultaneous analysis.

Main Results:

• Achieved simultaneous quantification of hundreds of proteins from mouse liver within one week of MS measurement time.
• Demonstrated accurate and rapid quantification of GST-tagged QconCAT internal standards.
• Showcased efficient separation and absolute quantification of complex mixtures using the improved 2D RP method.

Conclusions:

• The developed strategy significantly enhances the dynamic range and analytical sensitivity of SRM analysis.
• This approach increases the coverage of absolute quantification in whole proteome studies.
• The method offers a powerful tool for large-scale absolute protein quantification in complex tissues.