5 Peptide Bioanalysis Sample Preparation Challenges (and how to overcome them!)

Scientists that have worked with peptides understand that peptide quantification can be challenging. One of the keys to successful peptide analysis is thorough and selective sample preparation.

Sample preparation improves various aspects of chromatography and mass spectrometry analyses by utilizing solid phase extraction (SPE) to chemically separate different analytes and aid in removing the interfering matrix components in a sample.

Here, we’ll discuss five of the primary peptide sample preparation challenges including:

• Protein Binding
• Non-Specific Binding (NSB)
• Peptide Solubility
• Peptide Specificity
• Recovery

Protein Binding
In the case of Peptide analysis, thorough sample preparation can help address both protein binding and non-specific binding. One way to eliminate the binding of the peptide to proteins (or other matrix components) during your sample preparation is to dilute the plasma 1:1 with 4% H₃PO₄ or 5% NH₄OH. Depending on how strongly your peptides are bound to the proteins, you may need to take more aggressive steps like denaturation with Guanidine HCl, Urea or SDS, or even a 1:1 ratio PPT with ACN.

Non-Specific Binding
In order to address non-specific binding (NSB), an important consideration during sample preparation is the sample container. Peptides are notoriously sticky and may adhere to glass. Rather than using glass for sample preparation, try polypropylene or materials designed with high-performance surfaces for low binding of peptides and proteins. This will help reduce non-specific binding issues.

Additionally, peptides can be lost during sample preparation dry down and reconstitution steps due to adsorption onto the collection container, or failure to re-solubilize. This can be reduced by using a µElution format. Using a product such as a µElution plate allows you to skip evaporation and eliminates the need for reconstitution since the elution volume is generally only 25-50 microliters. Simply dilute the eluate 1:1 with water and inject.

Peptide Solubility
In order to prevent precipitation of your peptides, it is suggested that you limit the organic concentration to no more than 75%. You can also use modifiers to promote solubility, but it may require higher concentrations than what you have used with small molecules. Try a range from 1% up to 10% acid or base, such as TFA, FA, AA or NH₄OH.

Peptide Specificity
Peptide quantification requires highly sensitive and reproducible methods. In many cases, protein precipitation, very popular with small molecule bioanalysis, will not be clean enough for sensitive peptide methods. Specificity is gained by creating a sample preparation method that uses orthogonal cleanup approaches, both reverse-phase and ion exchange, to selectively capture your target peptide, while allowing you to wash away the unwanted matrix interferences. Then concentrate your peptide in a small elution volume that can be diluted with a little water and directly injected into the LC-MS system.

Recovery
Low or variable recoveries are generally symptoms of one of the four problems outlined above. When you experience low recoveries, revisit the steps you can take to reduce protein binding, non-specific binding, peptide solubility and increase peptide specificity in your sample cleanup protocol. One or all of these may be the key in helping improve your peptide bioanalytical method.

Additional information on Peptides and Sample Preparation

• 4-Step Workflow for Optimized Peptide Analysis
• Peptide and Protein Bioanalysis Boot Camp
• Lost Samples in the Container: Non-specific Binding and the Impact of Blocking Agents
• Navigating Challenges Associated with Bioanalysis of Therapeutic and Endogenous Peptides, a Six-Part Video Series

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