Fast and accurate absolute-quantification of proteins and antibodies using Isotope Dilution-Triple Quadrupole ICP-MS

Importance of the Topic

Absolute quantification of proteins and antibodies is crucial in life-science research, clinical diagnostics, and biopharmaceutical quality control. Traditional approaches often require compound-specific calibration standards, suffer from matrix biases, and involve lengthy sample preparation. Isotope dilution triple-quadrupole ICP-MS (ID-ICP-QQQ) targeting sulfur offers a universal, interference-free method for rapid, accurate protein analysis without the need for individual reference standards.

Objectives and Study Overview

• Assess the performance of an Agilent 8800 triple-quadrupole ICP-MS in MS/MS mass-shift mode with oxygen gas for sulfur isotope measurements.
• Apply single and double isotope dilution mass spectrometry to determine sulfur in NIST BSA 927e SRM, human immunoglobulin mixtures, and a trastuzumab monoclonal antibody.

Instrumentation

• Agilent 8800 ICP-QQQ with ORS3 collision/reaction cell operating in MS/MS mass-shift mode.
• Standard concentric MicroMist nebulizer, Peltier-cooled Scott double-pass spray chamber, quartz torch, nickel interface cones.
• O₂ cell gas for mass shift of ³²S⁺ and ³⁴S⁺ to SO⁺ product ions at m/z 48 and 50.
• Operating parameters: RF power 1550 W; nebulizer gas flow 0.25 L/min; dilution gas flow 0.85 L/min; O₂ flow 0.35 mL/min; data acquisition on shifted m/z values.

Key Results and Discussion

• BSA SRM (NIST 927e) quantification achieved recoveries of ~101% with RSD <0.35% and linearity R² = 0.99998 across varied sample loads.
• Formulation buffers (NaCl, PBS, sucrose, polysorbate 80, glycine, histidine) and their mixture produced mean sulfur recoveries of ~100% (RSD <0.65%), demonstrating excellent matrix tolerance.
• Double IDMS corrected for spike concentration and mass bias, ensuring high accuracy of sulfur measurements.
• Filtration experiments removed low-molecular-weight sulfur interferences (sulfate, methionine) while preserving protein-bound sulfur (>99% recovery).
• Intermediate precision tests between analysts and days yielded RSD ≤0.7% for both BSA and IgG samples.
• Trastuzumab antibody analysis delivered 97.8% recovery, confirming applicability to therapeutic monoclonal antibodies.

Benefits and Practical Applications

• High sensitivity and interference removal via MS/MS mass-shift chemistry.
• Absolute quantification without compound-specific standards.
• Fast workflow: microwave digestion and ID-ICP-QQQ analysis in a single day.
• Universal applicability to any sulfur-containing proteins or peptides in complex biological matrices.
• Ideal for proteomics, metallomics, biopharmaceutical QA/QC, and clinical studies.

Future Trends and Potential Applications

• Expansion to multi-element isotope dilution for comprehensive metalloprotein profiling.
• Automation and high-throughput digestion workflows for large-scale protein studies.
• Integration with chromatographic front ends for speciation and post-translational modification analysis.
• Application to advanced biotherapeutics, vaccines, and cell-therapy characterization.
• Development of novel reaction cell gases to extend interference removal to additional challenging elements.

Conclusion

The Agilent 8800 ICP-QQQ in MS/MS mode combined with isotope dilution offers a robust method for absolute quantification of proteins and antibodies through sulfur measurement. Its superior sensitivity, precision, and matrix tolerance eliminate the need for compound-specific calibration, providing a versatile tool for life-science research and biopharmaceutical quality control.

References

1. Anan Y., Hatakeyama Y., Tokumoto M., Ogra Y. Analysis of selenoproteins in rat serum by Triple Quadrupole ICP-MS. Agilent publication 5991-2750EN (2013).
2. Wind M., Wegener A., Eisenmenger A., Keller R., Lehmann W.D. Kinetic energy discrimination for elemental analysis. Angewandte Chemie Int. Ed. 42 (2003) 3425–3427.
3. Zhang H., Yan W., Aebersold R. Advances in quantitative proteomics. Curr. Opin. Chem. Biol. 8(1) (2004) 66–75.
4. Sugiyama N. Accurate measurement of selenium in twelve diverse reference materials using on-line isotope dilution with the 8800 Triple Quadrupole ICP-MS in MS/MS mode. Agilent publication 5991-0259EN (2012).
5. Balcaen L., Vanhaecke F., Woods G., Resano M. Accurate determination of sulfur in biodiesel using Isotope Dilution-Triple Quadrupole ICP-MS. Agilent publication 5991-2337EN (2013).