How to Perform Multiple-Specificity MS/MS in Peptide Ligands

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The best way to identify analytes in complex mixtures is through high-resolution mass spectrometry. This technology is also applicable to the identification of bioactive peptides. It has numerous applications in medicine, animal health, food, and agriculture. Here we describe how to perform a multiple-specificity study in peptide ligands using this technique.

For our example, we used high-resolution MS/MS to quantify five targets in human and mouse plasma. These peptides are from different proteases. We found that these peptides have antiallergic and antioxidant properties. They reduce production of reactive oxygen species and promote endogenous antioxidant defense systems. In addition, they have beneficial effects on bone health.

In order to find the most significant peptide, we performed a statistical analysis. The result was a two-sided unpaired t-test that revealed that the most significant peptide was not actually the one with the most mass, but the one with the largest p-value.

There are many flavors of peptide quantitation. One is the “top 10” method, whereby ten of the most abundant precursor ions are extracted from a sample. Another is the “pseudocount” method, where the number of pseudocounts is added using a substitution matrix. Finally, there is the “dynamic exclusion list” with an exclusion width of ten pppm. However, it is important to evaluate the robustness of the process. If a contaminant peptide peaks in an experiment, the results may be distorted.

Alternatively, we could have used a simpler method to compute the most significant peptide. To do this, we renormalized the expected frequency of amino acids in the peptide.


If you are interested in consuming peptides, it is imperative to consult a medical professional! If any action is taken as a result of this content, we cannot be held liable for any damages. Click on the icon to learn more about why a medical consultation is mandatory!

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