Why sequencing matters
Function is dictated by structure — and structure begins with the amino-acid sequence (the primary structure, read N→C terminus).
The story in one paragraph
Insulin was the first protein ever sequenced. Frederick Sanger won the Nobel Prize for it — but it took ~10 years, many people and 100 g of protein. Today one person sequences the same insulin in days. This module walks the classic workflow using a 14-residue teaching peptide, then lets you reconstruct its sequence yourself.
Meet insulin in 3D
Rotate/zoom with your mouse. Insulin's two chains are held together by disulfide bonds — the first thing you must break before sequencing. Highlight them below.
The 3-stage workflow
Above all else — purify it first. Then:
1 · Prepare the protein
Count the chemically different polypeptides · cleave the disulfide bonds · separate & purify each subunit · determine amino-acid composition.
Disulfides are broken to separate chains and to stop refolding. Two ways: performic acid (oxidises cysteine → cysteic acid) or reducing agents 2-mercaptoethanol / DTT (keeps the −SH reduced).
2 · Sequence the chains
Fragment each subunit into peptides <~50 residues · separate & purify fragments · sequence each fragment · repeat with a different cleavage method.
Why repeat? Even the best Edman chemistry reads only ~50 residues per run at ~98% efficiency, so long chains must be cut into overlapping pieces.
3 · Assemble the structure
Use overlapping fragments from the two digests to span the cleavage points · then locate disulfide bonds and any modified residues. You'll do exactly this in the puzzle.
Reading the N-terminus
Two jobs: identify the end residue, then read the chain one residue at a time.
End-group vs. stepwise
| Method | Reagent | Reads | Catch |
|---|---|---|---|
| Dansyl chloride | reacts with free amines (N-term + Lys) | N-terminal residue only | needs 6 M HCl to release it — destroys the rest of the chain |
| Edman degradation | phenyl isothiocyanate (PITC) | one residue per cycle, repeatedly | ~98% efficient → errors accumulate on long reads |
| Carboxypeptidase | enzyme | C-terminal residue(s) | timing/rate ambiguity if bonds cleave at similar rates |
Run the Edman cycle
Each cycle labels the N-terminal residue with PITC, cleaves it as a soluble PTH-amino acid, and leaves the shortened chain intact for the next round.
Cleavage reagents
The overlap trick only works if two reagents cut at different places. Know the specificities cold.
| Reagent | Type | Cleaves after… |
|---|---|---|
| Trypsin | endopeptidase | K, R (positively charged) — not before Pro |
| Chymotrypsin | endopeptidase | F, W, Y (bulky hydrophobic) — not before Pro |
| Thermolysin | endopeptidase | before I, M, F, W, V, L |
| Endopeptidase V8 | endopeptidase | E (glutamate) |
| Cyanogen bromide (CNBr) | chemical | M (methionine) |
Watch out — amino-acid composition
Composition is found by full hydrolysis. Acid hydrolysis (6 N HCl, 120 °C) destroys Trp, partly destroys Ser/Thr/Tyr, and converts Gln→Glu, Asn→Asp. Base hydrolysis spares Trp but destroys Cys/Ser/Thr/Arg.
Reconstruct the peptide
A 14-residue peptide was digested two ways. Drag each fragment along its lane to its correct start position. Overlaps between the two digests reveal the full sequence.
What you know
End-group analysis: N-terminus = Y (Tyr), C-terminus = K (Lys). CNBr cuts after M; trypsin cuts after K/R. Line the pieces up so overlapping residues match.
Check your recall
Five questions. Immediate feedback.