Simultaneous quantification of peptides and phosphopeptides by capLC-ICP-MS using the Agilent 8800/8900 Triple Quadrupole ICP-MS

Significance of the Topic

Accurate quantitation of peptides and phosphopeptides is essential in proteomics, clinical research and pharmaceutical development. Non-specific quantification based on heteroatom analysis (phosphorus and sulfur) provides a universal approach that avoids reliance on isotopically labeled standards. However, conventional ICP-MS methods suffer from poor detection limits and spectral interferences for these elements. The integration of capillary LC with a triple quadrupole ICP-MS in mass shift mode addresses these challenges, offering unprecedented sensitivity and interference removal.

Objectives and Overview of the Study

This study aimed to evaluate the performance of the Agilent 8800/8900 triple quadrupole ICP-MS coupled to capillary LC for simultaneous absolute quantitation of P- and S-containing peptides. Key goals included achieving the lowest possible detection limits, verifying interference elimination, and demonstrating quantification of model peptides and phosphopeptides using generic non-proteinaceous standards.

Methodology and Instrumentation

A reversed-phase capillary LC system (Agilent 1200 Series, Zorbax SB C18, 150×0.3 mm) was interfaced to the ICP-QQQ via a low-flow nebulizer system. Mobile phases contained 0.1% formic acid in water or acetonitrile with 10 ng/mL Ge as internal standard. The ICP-QQQ operated in MS/MS mass shift mode: Q1 selected m/z 32 for sulfur or m/z 31 for phosphorus, reactions with O2 in the octopole cell generated SO+ at m/z 48 and PO+ at m/z 47, and Q2 filtered out residual interferences. Calibration standards (0–200 ng/mL of P or S) and peptide mixtures were analyzed at 5 µL/min flow with a 1–2 µL injection volume.

Main Results and Discussion

Interference removal was confirmed by matching the observed 34S/32S isotope ratio to theoretical values within mass bias limits. In a high organic matrix, achieved detection limits were 0.6 ng/mL for P and 1.2 ng/mL for S; in LC operation they improved to 0.10 ng/mL P (6.6 fmol) and 0.18 ng/mL S (11 fmol), the lowest reported for LC-ICP-MS. Model peptides (ACTPERMAE, VPMLK) and phosphopeptides (LRRApSLG, KRSpYEEHIP) were quantified with excellent peak shapes and signal-to-noise ratios. Detection limits for analytes were slightly higher than standards due to broader chromatographic peaks and carbon-induced P sensitivity enhancement.

Benefits and Practical Applications

• Simultaneous absolute quantification of P- and S-containing peptides without labeled analogs.
• Lowest detection limits reported for peptide heteroatoms in LC-ICP-MS.
• Robust interference removal via MS/MS mass shift, enabling accurate analysis in complex matrices.
• Potential application in pharmaceutical quality control, environmental monitoring, and nanoparticle characterization.

Future Trends and Opportunities

Advances in ICP-QQQ design, such as reduced-background argon flow paths in the Agilent 8900, are expected to further lower detection limits, especially for sulfur. The ability to perform isotope dilution with accurate isotope patterns opens possibilities for absolute quantitation in high-throughput proteomics. Integration with nano-LC and automation could extend applications to clinical biomarker quantitation and environmental speciation studies.

Conclusion

The capillary LC coupled to an ICP triple quadrupole in MS/MS mode delivers unmatched sensitivity and interference-free measurement of phosphorus and sulfur in peptides. This methodology provides a universal, label-free strategy for quantitative proteomics and phosphoproteomics, with broad potential across research and industrial laboratories.

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