N-Terminal protein sequencing

Why study the N-Terminal of a protein?

Many biological processes require post-translational modifications to occur on a protein. For example, the majority of peptides hormones are produced as pro-hormones that need to be proteolytically cleaved to be active. Therefore, workflows that enable the identification of the N-Terminal of one or many proteins can become very interesting. Using a combination of chemical modifications, enrichment methods and mass spectrometry, we can produce a map of the "N-Terminome" of a sample. This workflow can be used to study the N-Terminus of pure proteins, or to identify variations in the N-Termini of every detectable protein in a sample.

General workflow

1. In N-Terminal mapping workflows, native proteins N-Termini are first protected with a chemical group, generally using amine dimethylation.
2. Proteins are digested with a protease, which generates non-native N-Temini.
3. The newly generated non-native N-Termini are labeled with a "targetable" chemical group.
4. Using a strategy that targets the second labeling reaction, the samples are depleted from every peptide whose N-Terminus was not protected with the first amine dimethylation reaction.
5. The result of this depletion will be a sample in which the only remaining peptides will be the ones who were protected with the first chemical reaction, which are the N-Termini peptides of every protein in the sample. These peptides will be analyzed by LC-MS/MS.

Data report

For N-Terminal sequencing of a pure protein

Data reports for N-Terminal sequencing include the MS and MS/MS spectra of the detected peptide and any additional information that may be needed.

Proteome wide N-Terminal sequencing

List of every detected and confirmed N-Terminal peptide. Any specific information that the project requires will also be included.