Peptide Mapping and Small Protein Separations with Charged Surface Hybrid (CSH) C18 and TFA-Free Mobile Phases

Importance of the Topic

Peptide mapping and the separation of small proteins are central to biopharmaceutical quality control, enabling detailed structural characterization and verification of therapeutic peptides and proteins. High-resolution reversed-phase LC coupled with sensitive MS detection is critical for identifying sequence variants, post-translational modifications, and process impurities. Avoiding strong ion-pairing agents such as TFA enhances mass spectrometric sensitivity, supporting robust peptide and small protein analysis in regulatory and research environments.

Study Objectives and Overview

This study evaluates the performance of a novel charged surface hybrid (CSH) C18 stationary phase for peptide mapping and the separation of polypeptides up to 12 kDa using trifluoroacetic acid-free mobile phases. Comparisons are made against conventional fully porous and superficially porous C18 columns under formic acid mobile phase conditions. The objectives include assessing peak capacity, selectivity, sensitivity, and applicability across a range of peptide and small protein analytes.

Methodology

A Waters ACQUITY UPLC H-Class Bio System coupled to a Xevo G2 Q-Tof mass spectrometer was used to analyze an enolase tryptic digest and a mixture of six polypeptides (1–12 kDa). Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. Gradients were optimized for peptide mapping and small protein separations. Analyses compared ACQUITY UPLC CSH130 C18 (130Å, 1.7 μm), BEH130 C18 (130Å, 1.7 μm), superficially porous C18 (100Å core–shell, 1.7 μm), and BEH300 C18 (300Å, 1.7 μm) columns at 0.3 mL/min and 40 °C, with UV (214 nm) and ESI-MS detection.

Instrumentation

• ACQUITY UPLC H-Class Bio System
• Xevo G2 Q-Tof Mass Spectrometer
• ACQUITY UPLC CSH130 C18, 1.7 μm, 2.1×150 mm column
• MassPREP Enolase Digestion Standard
• LCGC Certified Clear Glass Qsert Vials

Main Results and Discussion

• The CSH130 C18 column achieved a peak capacity of 532 for the enolase digest with 0.1% formic acid, outperforming BEH130 C18 (399) and superficially porous C18 (405).
• TFA in mobile phases caused an order-of-magnitude drop in MS sensitivity, whereas formic acid preserved sensitivity without compromising peak shape on CSH130 C18.
• CSH130 C18 provided unique selectivity driven by low-level positive surface charge, altering elution order of histidine-containing peptides and improving resolution of critical peptide pairs.
• For polypeptides up to 12 kDa, CSH130 C18 delivered superior peak shape across most species, including insulin analogs, compared to pores of 100–300Å. BEH300 C18 better resolved the largest proteins (8.6–12.4 kDa), while superficial particles underperformed for larger analytes.

Benefits and Practical Applications

• High peak capacity enhances detection of low-abundance peptides and minor variants.
• TFA-free mobile phases ensure optimal ESI-MS sensitivity and quantitative accuracy.
• Charged surface hybrid chemistry offers alternative selectivity for challenging peptide maps.
• Suitable for a broad molecular weight range, from small tryptic peptides to early-stage biotherapeutics up to ~10 kDa.

Future Trends and Opportunities

The integration of charged surface stationary phases with advanced UPLC-MS platforms will support deeper proteome coverage and higher throughput. Future work may explore pore-size combinations, surface chemistries, and gradient optimizations for large biomolecules, intact protein analysis, and combinatorial screening of column chemistries to tailor selectivity for complex biologics.

Conclusion

The CSH130 C18 column coupled with formic acid mobile phases significantly improves peptide mapping and small protein separations for LC-ESI-MS applications. Its high peak capacity, enhanced MS sensitivity, and unique charge-driven selectivity make it a versatile choice for routine biopharmaceutical analysis and method development.

References

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