Quantification of protein phosphorylation by microLC-ICP-MS

Area of Science:

• Analytical Chemistry
• Biochemistry
• Proteomics

Background:

• Accurate quantification of protein amount and phosphorylation extent is vital across research disciplines.
• Current methods for phosphoprotein analysis can be challenging and lack precision.

Purpose of the Study:

• To introduce and validate capillary liquid chromatography-inductively coupled plasma-mass spectrometry (LC-ICP-MS) for phosphoprotein quantification.
• To establish a method for determining both protein abundance and phosphorylation degree.

Main Methods:

• Utilized capillary LC-ICP-MS for sensitive detection of phosphorus and sulfur in proteins.
• Employed sulfur as an internal standard for protein quantification and phosphorus for phosphorylation assessment.
• Applied isotope dilution analysis with a 34S-Spike for highly accurate absolute protein quantification.
• Demonstrated applicability to isolated proteins, protein digests, and complex cellular extracts after SDS-PAGE and enzymatic digestion.

Main Results:

• Demonstrated the capability of LC-ICP-MS to quantify phosphoproteins and their phosphorylation degree.
• Established a method to determine phosphorylation stoichiometry by comparing phosphorus to sulfur abundance.
• Achieved high accuracy in absolute protein quantification using isotope dilution mass spectrometry.
• Showcased the method's versatility for various sample types, including complex biological mixtures.

Conclusions:

• Capillary LC-ICP-MS offers a robust and accurate approach for phosphoprotein analysis.
• The method enables precise determination of phosphorylation stoichiometry and global/tissue-specific phosphorylation degrees.
• This technique provides a valuable tool for advancing research in proteomics and cell signaling.