Protein Sequencing Methods
Idea creds @Delphinidin!
There are three main protein sequencing techniques: Sanger protein sequencing, Edman degradation, and the most efficient/most widely used today, peptide mass spectrometry. All 3 methods sequence from the amino to carboxyl ends.
In Edman degradation, the protein is sequenced from the N terminal to C terminal. The N terminal amino acid is labeled by Edman's reagent in alkaline (basic) conditions. Applying heat and acidic conditions breaks this labeled amino acid off the chain. The amino acid is then identified through high-performance liquid chromatography (HPLC) which filters the entity of interest based on its affinity to a particular substrate (kind of like a magnetic pull towards specific things, but everything else is discarded). The retention time and other behaviors of the amino acid in the HPLC procedure allow it to be visually matched to a known amino acid chromatogram. (Retention time is the amount of time the substance sticks to the column, and the chromatogram is the diagram generated from this experiment.) Putting together the data from the runs for each amino acid helps us build the sequence of the original protein, from the N to C terminus.
There are many different kinds of mass spectrometry, but here I will go over ESI-TOF mass spectrometry because that's the one I work with and am most familiar with. ESI means electrospray ionization, and TOF means time of flight. Basically, you shoot your protein sample into the machine and it sprays the sample into droplets so minute that they start to act like gases (like they can fly around). So these particles, now parent ions, get smashed in a strategic way, and both these daughter ions' and parent ions' times of flight through the machine are measured to determine mass to charge (m/z) ratio, which helps determine the identity of the original parent ion and therefore protein. Essentially, we have broken the original proteins into manageable identifiable pieces, and we identified the pieces, and put them back together to identify the original protein.
Sanger protein sequencing is basically the above two combined and antiquated; it was the OG protein seq technique before MS. It actually uses Edman degradation as part of the process. Like mass spec, you chop the protein up into manageable peptides using an enzyme, except in mass spec, the ionization procedure chops the protein up even more than just into peptides. Then, each peptide fragment is analyzed using Edman degradation; this is the Sanger method of protein sequencing.
Here are some helpful videos:
More on Sanger: https://youtu.be/OeJqXWChfqk
More on Edman: https://youtu.be/GUhZoNTCCPo?si=rpAC6iNs-mOLHX27
More on HPLC graphs and retention time etc: https://youtu.be/qXmSb6Xwr5k
More on mass spec: https://youtu.be/hSirWciIvSg
There's also Sanger DNA seq: https://www.sixfootscience.com/brain-snips/how-dna-sequencing-works-sanger-and-ngs
