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

Significance of the topic

Quantification of phosphorus and sulfur in peptides is essential for proteomics and phosphoproteomics, providing accurate measurements of phosphorylation and sulfur-based modifications in proteins. Conventional quadrupole ICP-MS techniques face challenges from polyatomic interferences and high detection limits for P and S. The implementation of a triple quadrupole ICP-MS in MS/MS mass shift mode overcomes these limitations, delivering enhanced sensitivity and specificity.

Objectives and study overview

This study demonstrates the simultaneous absolute quantification of phosphopeptides and sulfur-containing peptides by coupling capillary LC with the Agilent 8800 Triple Quadrupole ICP-MS. Key aims include achieving ultralow detection limits for P and S, validating interference elimination, and employing generic heteroatom standards for non-species specific quantification.

Methodology

• Capillary LC separation using an Agilent 1200 Series system with Zorbax SB C18 column (150 × 0.3 mm, 5 µm), 5 µL/min flow, gradient from 1% to 60% acetonitrile (0.1% formic acid).
• Calibration with bis(4-nitrophenyl) phosphate (BNPP) and methionine standards at 0–200 ng/mL P or S, using 10 ng/mL Ge as internal standard.
• MS/MS mass shift operation: Q1 selects m/z 31 or 32, ORS cell reaction with O2 generates PO+ (m/z 47) and SO+ (m/z 48), Q2 filters oxide ions to remove interferences.
• Interference verification by matching the 34/32 S isotope ratio and confirming mass discrimination factors consistent with literature values.

Instrumentation

• Agilent 1200 Series capillary LC system
• Agilent Zorbax SB C18 capillary column
• Agilent capLC interface kit (G3680A) with total consumption nebulizer
• Agilent 8800 Triple Quadrupole ICP-MS operated in MS/MS mass shift mode with oxygen reaction gas and Ge internal standard
• Syringe pump for continuous organic matrix introduction

Key results and discussion

• Achieved detection limits of 6.6 fmol for phosphorus (0.10 ng/mL) and 11 fmol for sulfur (0.18 ng/mL), the lowest reported by LC-ICP-MS to date.
• Excellent linearity (RSD < 4%) over 25–200 ng/mL for both elements.
• Effective removal of interferences confirmed by correct isotope ratios and oxide ion filtering.
• Simultaneous analysis of phosphopeptides (LRRApSLG, KRSpYEEHIP) and sulfur peptides (ACTPERMAE, VPMLK) showed sharp peaks and high signal-to-noise ratios; slight sensitivity enhancement for P in organic-rich eluents noted.

Benefits and practical applications

This approach enables absolute quantification of peptides using generic heteroatom standards, eliminating the need for isotope-labeled compounds. It offers robust application in high-throughput proteomics, pharmaceutical QA/QC, environmental pesticide monitoring, and characterization of P- and S-containing nanomaterials.

Future trends and potential applications

• Adoption of next-generation ICP-QQQ instruments (e.g., Agilent 8900) with reduced elemental background for further improved detection limits.
• Extension to multi-heteroatom and molecular speciation analyses by coupling with high-resolution mass spectrometry.
• Development of automated on-line sample preparation workflows for clinical and industrial throughput.
• Standardization of protocols for regulatory compliance and diagnostic applications in biopharma.

Conclusion

The integration of capillary LC with Agilent 8800 ICP-QQQ in MS/MS mass shift mode achieves unparalleled sensitivity and interference-free quantification of P and S in peptides. This element-specific platform supports absolute proteomic analyses without species-specific standards and promises broad application across scientific and industrial fields.

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