A Database of Accurate Electrophoretic Migration Patterns for Human Proteins.

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Native molecular weight (MW) is crucial for protein characterization.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is widely used for protein MW determination and quantification.
• Current SDS-PAGE methods suffer from poor reproducibility and inaccurate MW measurements, impacting antibody validation and western blot reliability.

Purpose of the Study:

• To establish a database of reference MWs measured by SDS-PAGE to improve reproducibility and accuracy.
• To develop a method for accurate and reproducible MW determination using mass spectrometry and internal calibration.
• To provide a freely accessible web resource for researchers to troubleshoot western blot experiments and characterize proteoforms.

Main Methods:

• Utilized mass spectrometry as an orthogonal detection method to analyze electrophoretic migration patterns.
• Acquired migration data for approximately 10,000 human proteins across five common cell lines.
• Applied robust internal calibration of migration to determine accurate and reproducible molecular weights.

Main Results:

• Generated accurate and reproducible molecular weights for a large set of human proteins.
• Enabled merging of replicates for increased accuracy and comparison across different cell lines.
• Identified structural factors influencing protein migration and recapitulated known post-translational modifications and differential splicing.

Conclusions:

• The developed database provides accurate reference MWs, enhancing the reliability of SDS-PAGE and western blot experiments.
• The resource facilitates troubleshooting of western blot experiments and aids in the characterization of human proteoforms.
• The findings highlight the utility of mass spectrometry-coupled SDS-PAGE for proteomic analysis and hypothesis generation.