Enhanced Universal Quantification of Biomolecules Using Element MS and Generic Standards: Application to Intact...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Absolute quantification of biomolecules, especially proteins and post-translational modifications, is challenging without specific standards.
• Existing methods require tailored standards, limiting broad applicability and increasing costs.

Purpose of the Study:

• To develop and optimize a robust method for absolute biomolecule quantification using HPLC-ICP-MS without specific standards.
• To enhance the previously introduced CH4-based plasma strategy for improved safety and efficiency.
• To demonstrate the method's capability for accurate protein quantification, elemental speciation, and analysis of complex biological samples.

Main Methods:

• Optimization of CO2 addition to plasma in HPLC-ICP-MS for stable signal response factors across multiple elements (S, P, As, Se, Br, I).
• Comparison of accuracy and precision for S-based absolute protein quantification using generic internal and external standards.
• Application of the method for high-sensitivity analysis of snake venom toxins and simultaneous P and S speciation in phosphoproteomics.

Main Results:

• A constant signal response factor (<6% error) was achieved for six elements under compromised conditions using CO2 addition.
• Demonstrated high-sensitivity analysis of toxins in snake venoms, showcasing real sample analysis potential.
• Achieved simultaneous absolute protein amount and phosphorylation degree determination for intact β-casein and its isoforms with unprecedented low detection limits (<1 fmol protein).

Conclusions:

• The optimized CO2-based HPLC-ICP-MS strategy offers a reliable and sensitive approach for absolute biomolecule quantification without specific standards.
• The method enables accurate multielemental speciation, particularly valuable for phosphoproteomics.
• This advancement significantly lowers detection limits for protein quantification, opening new avenues in biochemical analysis.