Simultaneous Quantification of Peptides and Phosphopeptides by capLC-ICP-MS

Significance of the Topic

The quantification of peptides and phosphopeptides is critical in proteomics, pharmaceutical research, and clinical studies. Conventional LC-MS/MS methods require isotopically labeled standards for each target analyte. In contrast, ICP-MS leverages element-specific detection, enabling generic quantitation of phosphorus and sulfur containing species without compound-specific labels. This approach promises simplified workflows and broad applicability, provided polyatomic interferences and high detection limits can be overcome.

Objectives and Study Overview

This study evaluated the performance of an Agilent 8800 triple-quadrupole ICP-MS (ICP-QQQ) coupled to capillary LC for simultaneous non-species-specific absolute quantification of P and S in peptides. Generic standards—bis(4-nitrophenyl) phosphate (BNPP) for P and methionine for S—were used to establish calibration curves. The method was applied to model phosphopeptides (LRRApSLG, KRSpYEEHIP) and sulfur-containing peptides (ACTPERMAE, VPMLK) to assess sensitivity, linearity, interference removal, and applicability to peptide analysis.

Methodology and Instrumentation

• Sample Preparation: Calibration standards at 0, 25, 50, 100, 200 ng/mL elemental P or S in 1% acetonitrile mobile phase; phosphopeptide and sulfur-peptide mixtures prepared at ~100 ng/mL element plus generic standards.
• Chromatography: Agilent 1200 Series capillary LC with Zorbax SB C18 column (150×0.3 mm, 5 µm); water (A) and acetonitrile (B) containing 0.1% formic acid and 10 ng/mL Ge internal standard; flow 5 µL/min; gradient from 1% to 60% B over 35 min; injection 1–2 µL.
• ICP-QQQ Operation: Agilent 8800 triple-quadrupole ICP-MS; O2 reaction gas (0.35 mL/min in cell; 8:2 Ar:O2 nebulizer gas at 80 mL/min); MS/MS mass-shift mode with Q1/Q2 settings for 32S→48SO+ and 31P→47PO+; integration time 150 ms; data acquisition via MassHunter.

Main Results and Discussion

• Detection Limits: Initial DLs of 0.6 ng/mL P and 1.2 ng/mL S in organic matrix improved to 0.10 ng/mL P (6.6 fmol) and 0.18 ng/mL S (11 fmol) under capLC-ICP-QQQ, the lowest absolute limits reported for LC-ICP-MS.
• Linearity and Precision: Calibration curves from 25 to 200 ng/mL yielded R2>0.999 and RSD<4%.
• Interference Removal: MS/MS mass-shift effectively removed polyatomic interferences, validated by accurate 34S/32S isotope ratios matching theoretical values after bias correction.
• Peptide Analysis: Phosphopeptides and sulfur-peptides were baseline separated with high signal-to-noise ratios; carbon matrix effects slightly enhanced phosphorus sensitivity.

Benefits and Practical Applications

This capLC-ICP-QQQ approach enables molecule-independent absolute quantitation without the need for compound-specific isotope labels. It is particularly suited to proteomics workflows, pharmaceutical and biopharma QC, trace environmental and food analyses, and biomarker screening where generic elemental detection streamlines method development.

Future Trends and Applications

Emerging instrument designs, such as the Agilent 8900 ICP-QQQ with low-sulfur components, will further reduce elemental backgrounds and lower detection limits. Integration with nanoLC, automated sample preparation, isotope-dilution strategies, and multiplexed assays could broaden applications in phosphoproteomics, metabolomics, and high-throughput biomarker profiling.

Conclusion

The combination of capillary LC with triple-quadrupole ICP-MS in MS/MS mass-shift mode achieves unprecedented sensitivity and interference-free quantification of P- and S-containing peptides. This versatile platform simplifies elemental assays and offers robust performance for diverse analytical challenges.

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