Edman degradation
Read a protein's N-terminal sequence one residue at a time.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
- Why a 1950 method is still on the service list
- The chemistry, one residue at a time
- What the readout actually is
- Why the read length is limited
- A blocked N-terminus stops it dead
- It needs far more material, and it needs it pure
- Things Edman can tell you that mass spectrometry finds awkward
- Key terms
- When to use it
1Why a 1950 method is still on the service list
Mass spectrometry has replaced Edman degradation for almost everything it used to do. It is more sensitive by two or three orders of magnitude, it reads thousands of proteins at once, and it handles mixtures and modifications that stop Edman chemistry dead.
2The chemistry, one residue at a time
Phenyl isothiocyanate couples to the free α-amino group at the N-terminus under mildly basic conditions. Anhydrous acid then cleaves the first peptide bond, releasing only the derivatised N-terminal residue as an anilinothiazolinone and leaving the rest of the chain intact with a new free N-terminus, one residue shorter.
3What the readout actually is
The data are a stack of HPLC chromatograms, one per cycle, each showing every PTH-amino acid present plus the reagent by-products. Calling a residue is not reading the tallest peak, because by the middle of a run the previous residue is still coming off and every amino acid is present as background.
4Why the read length is limited
Repetitive yield
Each cycle loses a few percent of the chains: washout, side reactions, chains that cannot couple again. At 93 to 95% per cycle the signal halves roughly every ten to twelve cycles, and it never stops falling.
Lag
A small fraction of chains fails to couple or cleave in each cycle and falls one residue behind. The out-of-phase signal accumulates, so every cycle carries more of the previous residue alongside the current one.
Rising background
Acid occasionally cleaves inside the chain, creating new N-termini that start sequencing from random positions. That background of every amino acid grows with cycle number while the real signal shrinks.
Weak residues
Serine and threonine dehydrate during conversion and give small, split peaks; tryptophan and histidine recover poorly; cysteine gives nothing unless it was alkylated first. A weak residue next to a strong neighbour's lag is the classic miscall.
Put those four together and the useful read from a good sample is usually 15 to 30 residues, occasionally more. The number that decides it is the amount of free N-terminus in cycle 1, which is why the yield plot (amount of the called residue against cycle, on a log scale) is the first thing to look at.
5A blocked N-terminus stops it dead
Edman needs a free α-amine. If anything sits on it, the first coupling never happens and every cycle reads background.
6It needs far more material, and it needs it pure
A routine Edman sample is tens of picomoles: about a microgram of a 25 kDa protein, against the femtomoles a mass spectrometer needs. Only around half of what is loaded is seen in cycle 1.
7Things Edman can tell you that mass spectrometry finds awkward
- Leucine and isoleucine are separate PTH peaks, so Edman distinguishes them; they have identical masses and standard MS cannot.
- The answer is positional and absolute: residue 1 is whatever came off in cycle 1. There is no inference from a database and no dependence on what was predicted.
- A ragged N-terminus from imprecise processing shows up directly as two or more sequences offset by a residue or two, with their relative amounts.
- A blank cycle is information: unmodified cysteine, a glycosylated asparagine or serine, or a phosphorylated residue all leave a gap where the standard peak should be.
Key terms
Practise these as flashcards →The Edman reagent. Couples to a free N-terminal amine under basic conditions so that acid can then cleave off just that one residue.
The phenylthiohydantoin derivative of the released residue, identified by its HPLC retention time against a standard mixture.
The amount of the first residue actually detected in cycle 1, usually 30 to 60% of the protein loaded. It sets how far the read can go.
The fraction of signal kept from one cycle to the next, typically 90 to 95%. Fitted from the slope of a log plot of called residue against cycle.
Out-of-phase signal from chains that failed to advance in an earlier cycle, so the previous residue keeps appearing alongside the current one.
An N-terminal amine carrying an acetyl, formyl or pyroglutamate group, which cannot couple to PITC; the run gives background only.
The enzyme that removes an N-terminal pyroglutamate, exposing residue 2 for sequencing. It does nothing for acetylated N-termini.
Protein transferred from an SDS-PAGE gel onto polyvinylidene difluoride, the standard support for sequencing a single band. Transfer in an amine-free buffer such as CAPS.
When to use it
- To confirm the N-terminus of a recombinant protein, including whether a signal peptide, tag or initiator methionine was removed where predicted.
- To locate a proteolytic or processing cleavage site on a purified fragment, without depending on a database search.
- To get a first stretch of sequence from a protein of an organism without a sequenced genome, to design primers or identify it.
- To distinguish leucine from isoleucine, or to see a ragged N-terminus and the proportion of each form directly.
- For regulatory characterisation of a biologic, where N-terminal sequence is often specified as part of identity testing.
Compared with alternatives
Far more sensitive and handles mixtures and blocked N-termini, but identifies by database match; choose it for anything except reading an N-terminus from first principles.
What you need when Edman gives a blank cycle or a blocked N-terminus, because mass spectrometry measures the modification rather than stopping at it.
The usual way to get a single species for sequencing: run, blot to PVDF, stain and excise the band.
Produces the proteins whose processing Edman is most often asked to confirm: signal peptides, tags, initiator methionine.
Confirms the DNA you designed; Edman confirms the N-terminus the cell actually made from it, which is not always the same thing.