Since a protein digest may have hundreds of peptides, resolution is critical for confidence in your results. UPLC Peptide Analysis Solutions deliver unparalleled resolution and sensitivity in less time when compared to traditional HPLC methodologies.

Waters scientists considered every aspect of peptide analysis — from chemistries, instrumentation, software and methodology to usability, documentation, application support and remote services — to create fit-for-purpose system solutions for peptide analysis and peptide mapping. The results are better resolution of complex protein digests, exceptional glycopeptide resolution, and higher throughput.

Improve peak shape and reproducibility
Achieving optimal liquid chromatography-based analysis of biotherapeutics is often challenging due to non-specific interactions between internal instrument surfaces and biomolecules. Such interactions can contribute to recovery issues, retention time shift and poor peak shapes. Traditional solution based on passivation and conditioning runs often brings additional complexities to experiments and is a risk factor in achieving optimal repeatability and intermediate precision.

Waters' ACQUITY PREMIER solution featuring MaxPeak  high performance surface is designed to minimize interactions between instrument internal surface and biomolecules, improving recovery, retention time consistency, and peak shapes. The overall solution helps to better control variability risks, enhance decision making, and improve lab productivity.

Keys to Successful Peptide Analysis

Peptide mapping is an important technique for the comprehensive characterization of biopharmaceutical products. In a peptide map, each peptide fragment must be resolved into a single peak, which is a significant chromatographic challenge due to the inherent complexity of protein digests.

In addition to the large number of peptide fragments that are generated from the enzymatic digestion of a protein, the number of alternative peptide structures (post-translational modifications, oxidations, etc.) can be equally difficult. Achieving adequate resolution in peptide maps is a tedious process. Good methods often have long run times and may not be as rugged as desired. These factors have all been addressed with the UPLC® Peptide Analysis Solution.

With this specialized system configuration, you benefit from:

• Enhanced resolution, reproducibility, and sensitivity
• Qualitative and quantitative analysis of variant peptides with chromatographic resolution and undistorted peak shape at extreme molar ratios
• Wide dynamic range ensuring that peptides at extreme molar ratios are detected even at the lowest limits of quantitation
• Flexibility in peptide mapping, essential for characterizing proteins
• Choices for method development with columns that are quality control tested and certified using a peptide digest to ensure reproducibility

Significantly Higher Resolution

UPLC^® provides notable improvements in peptide mapping when compared to HPLC. Exceptional resolution is possible because separations obtained through the use of small particles result in narrower peaks. Just as significant, the surface chemistry of our Peptide Separation Technology packing material provides exceptional peak shape for a wide range of peptide structures.

This remarkable column performance can only be achieved with the optimized instrumentation of the ACQUITY UPLC^® System, which features reduced system volumes, minimal detector band-broadening and accelerated data acquisition rates required to preserve high-efficiency separations, while maximizing sensitivity.

Comparison of HPLC vs. UPLC resolving power

UPLC can provide twice the resolving power, as shown in the comparison of the separation of a tryptic digest of yeast alcohol dehydogenase by HPLC and UPLC.

Qualitative and Quantitative Analysis of Variant Peptides

All the elements of the UPLC Peptide Analysis Solution contribute to the successful qualitative detection and quantitative measurement of trace peptides:

• The performance of ACQUITY UPLC based fluidics ensures retention time reproducibility
• High-resolution separations of both large and small peaks are essential for accurate integration
• Peptide Separation Technology particles deliver chromatographic resolution and undistorted peak shape at extreme molar ratios
• The system’s advanced optical and mass detection technologies have a wide dynamic range ensuring that peptides, at these extreme molar ratios, are detected even at the lowest limits of quantitation

Deamidation of the peptide containing Asn and Gly, T-16, shifts the retention time and resolves into three unique chromatographic peaks

A tryptic digest of bovine hemoglobin was spiked with the peptide mixture as a surrogate to represent modified peptides and separated with a Peptide Separation Technology Column. The surrogate peptide concentrations were 0.2 to 2% of the digest concentration on a molar basis. The top panel shows the peptide map of bovine hemoglobin with peptide standards spiked at 2% molar basis. A total injection on-column of 200 pmol of hemoglobin digest was made. The bottom panel focuses on the elution position of the surrogate. Thus a small trace peptide can be identified and quantitated, as shown, down to a 0.2% level. To achieve this sensitivity resolution, peak shape must be maintained over a wide mass load.