Enhancing Phosphopeptide Quantitation using PREMIER Peptide CSH C18 Columns

Significance of the Topic

Phosphorylation is a critical post-translational modification that regulates cell signaling and many biological processes. Accurate quantitation of phosphopeptides is essential in proteomics, drug discovery, and kinase inhibitor studies. However, acidic peptides and phosphopeptides tend to adsorb to metal surfaces in LC systems, causing sample loss, poor reproducibility, and compromised MS data quality.

Study Objectives and Overview

This application note evaluates PREMIER Peptide CSH C18 columns featuring MaxPeak High Performance Surface technology for enhanced recovery and reproducibility of phosphorylated peptides. Conventional columns were compared to PREMIER hardware using an equimolar peptide mixture, and quantitative performance was further assessed with a doubly phosphorylated 5-FAM labeled peptide via spectrofluorometric detection.

Methodology and Instrumentation

Three conventional ACQUITY UPLC Peptide CSH C18 columns were compared to three ACQUITY PREMIER Peptide CSH C18 columns under identical conditions. Key instrument and chromatographic parameters included:

• LC system: ACQUITY PREMIER with MaxPeak HPS flow path
• MS system: Xevo G2-XS QTof in positive ESI mode
• Column dimensions: 2.1 × 50 mm, 1.7 µm, 130 Å
• Mobile phase A: 0.1 percent formic acid in water
• Mobile phase B: 0.09 percent formic acid in acetonitrile
• Flow rate: 0.2 mL/min; column temperature: 60 °C; sample temperature: 4 °C
• Detection: UV at 214 nm; spectrofluorometer excitation 443 nm, emission 516 nm
• Data systems: Empower 3, MassLynx, and UNIFI

Sample mixtures included angiotensin I, enolase T37, and a doubly phosphorylated insulin receptor peptide. Recovery and peak capacity were monitored by UV, and metal adduct formation was evaluated by MS.

Main Results and Discussion

PREMIER columns delivered high recovery and consistent performance from the very first injection, while conventional columns required extensive conditioning and still achieved only partial recovery of the doubly phosphorylated peptide. Key findings:

• Unconditioned conventional columns showed almost no recovery of doubly phosphorylated peptide on initial injections, versus nearly full recovery with PREMIER columns.
• Post-conditioning recovery on conventional hardware reached only 39 percent of PREMIER performance.
• MS spectra from conventional columns exhibited abundant iron adducts across peptide charge states, whereas PREMIER columns reduced metal adducts by 80 to 90 percent.
• PREMIER columns favored lower charge state ions, improving spectral clarity and sensitivity.
• Spectrofluorometric quantitation of a 5-FAM labeled phosphopeptide confirmed average recoveries above 93 percent on PREMIER hardware versus 60 to 66 percent on conventional systems.

Benefits and Practical Applications

The use of MaxPeak HPS surfaces in PREMIER columns offers:

• Substantially improved recovery of acidic and phosphorylated peptides without complex mobile phase additives or chelators
• Minimal or no column conditioning required
• Enhanced reproducibility and method robustness for low-level quantitation
• Cleaner mass spectra with minimal metal adducts, leading to more reliable MS identification and quantitation

Future Trends and Opportunities

Advances in inert column surfaces will expand applications beyond phosphoproteomics to other negatively charged analytes such as sulfonated peptides, acidic metabolites, and nucleotides. Integration of these surfaces with high-throughput platforms and miniaturized systems may further improve sensitivity. Continued development of surface chemistries tailored to specific analyte classes promises to enhance quantitative accuracy in complex matrices.

Conclusion

PREMIER Peptide CSH C18 columns with MaxPeak HPS technology effectively mitigate metal-analyte interactions, yielding superior recovery, reproducibility, and MS data quality for phosphorylated peptides. This hardware solution streamlines phosphopeptide analysis by eliminating the need for harsh mobile phase modifications and extensive conditioning, thereby improving the robustness and sensitivity of bioanalytical assays.

References

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