Optimizing your peptide mapping workflow

Peptide maps provide a more comprehensive analysis than intact protein analysis, delivering identification of proteins and their variants. They help to determine post-translational modifications (PTMs) and their locations, and confirm protein sequences. Peptide mapping is conducted using reversed-phase chromatography, often linked to mass spectrometry. This article follows on from our previous piece on the topic to look directly at optimizing some simple areas of your peptide mapping workflow to achieve the best possible results.

Improving efficiency

Column choice can have a huge impact on your results, and the time needed to fully resolve the protein. So, make sure that you consider column specifications carefully for the best results. Here are a few things to think through:

  • Length of the column: longer columns are usually better for fully resolving more complex peptide maps and providing higher resolution. Typically, it is best to start with a 150 mm column
  • Use a 2.1 id column to optimize mass spectrometry analytical sensitivity
  • Columns with smaller pore sizes, such as the Agilent AdvanceBio peptide mapping columns, are ideal for small protein fragments and peptides.

For a detailed review of column choice read our previous article.

Small changes can have big effects

For many, the main goal when looking to improve peptide mapping workflow is to minimize the analysis time, while retaining selectivity and high resolution. You can optimize your methods in a few ways to improve your results – let’s consider the gradient conditions, temperature, and gradient steepness, as examples.

Gradient conditions

A low pH ACN gradient is always recommended for peptide separation as it allows for the separation of a wide range of peptide types and structures. Low pH ensures protonation of residual silanols in the column, preventing undesirable interactions with basic amino acids that lead to poor peak shapes. A low pH gradient also helps to denature the peptide, improving retention and resolution. Importantly, it will provide narrower peaks in the separation due to the lower viscosity of the mobile phase.


Changing the temperature is a powerful way to alter selectivity and improve your peptide mapping workflow. It should be one of the first things to consider when looking to improve band spacing, as it is much more convenient and efficient than changing the mobile phase or column. An initial temperature of between 30 and 60 °C is recommended. However, the optimum temperature for a particular mapping separation will depend on many factors based on digestion type and composition.

Elevating the temperature produces narrower peaks, which can be seen in the chromatograph. The myoglobin tryptic digest was first separated at 30 °C, the second run was performed at 60 °C. The chromatograph illustrates changes in peak shape and position. The second trace shows improved separation between peaks 1, 2, and 3 and better selectivity between peaks 4 and 5.

Gradient steepness

Changing the gradient steepness can also dramatically improve band spacing and affect selectivity of the peptide mapping separation. Gradient steepness can be decreased by increasing the run time or by keeping the run time the same and shortening the elution time. It can be increased by keeping the run time constant and changing the flow rate.

The second set of chromatographs shown here demonstrate selectivity changes resulting from varying gradient steepness. Using a myoglobin tryptic peptide digest, a steep gradient run time of 15 minutes was compared to longer gradient run time of 40 minutes. Both separations were maintained at a flow of 0.6 mL/min at 50 °C. A comparison of the chromatograms – and identifying the same peaks (asterisks) in each separation – confirms numerous changes to band spacing, peak counts, and peak shape.

Partnering with you to get great results

This article looks at just a few considerations to optimize your peptide mapping workflow. For a detailed look at the sample preparation or protein digestion process, check out Agilent’s how-to guide.

For more information or assistance with peptide mapping separation, Agilent CrossLab service plans are available. Agilent CrossLab provide rapid onsite support, multivendor instrument service, remote instrument diagnostics, and more. The expert engineers strive to provide insight improving efficiency from one instrument to the entire lab.

Discover Agilent’s full range of peptide mapping resources here

For more information on any biopharma queries you may have, make sure to look at our AdvanceBio Expert blog series: http://www.chromnews.com/category/advancebio-expert/

For Research Use Only. Not for use in diagnostic procedures.