Accepted answer
Your intuition is correct, the coverage figure is honest and nearly meaningless, and the cause is a structural feature of tirzepatide: it contains no arginine, and its only unblocked lysine is at position 16.
Why trypsin gives two fragments
Tirzepatide is 39 residues, Aib at 2 and 13, acylated Lys20, C-terminal amide. Trypsin cleaves after Lys and Arg. There is no Arg. Lys20 bears the AEEA-AEEA-gamma-Glu-C20-diacid side chain on its epsilon-amine, so trypsin will not touch it. That leaves Lys16, followed by Ile17, which is not proline, so it cleaves.
| Fragment | Residues | Length | Monoisotopic mass (Da) | Observed as |
|---|
| T1 | 1-16 | 16 | 1807.85 | 2+ at 904.93 |
| T2 | 17-39 | 23 | 3020.68 | 2+ at 1511.35, 3+ at 1007.90 |
Those two masses sum correctly: 1807.85 + 3020.68 - 18.01 = 4810.52, which is the monoisotopic mass of intact tirzepatide. So the report is internally consistent and the arithmetic checks out. Coverage is genuinely 39 of 39.
Why 100% coverage is the wrong metric
Coverage tells you which residues appeared inside some identified peptide. It does not tell you how precisely a defect could have been located, and localisation is the entire reason to run a map. The useful way to think about it is average fragment length, or more directly, the number of residues over which a modification would be indistinguishable.
On this report, a mass shift anywhere in T2 is localised to a 23-residue window that contains the acylation site, the Trp, all three prolines and the C-terminal amide. A +16 on that fragment could be Trp25 oxidation or several other things. A -18 could be Ser dehydration at 32, 33 or 39. You have not localised anything; you have subdivided the molecule into two halves.
Compare a Glu-C digest. Glu-C in phosphate buffer cleaves after both Glu and Asp; tirzepatide has Glu3, Asp9 and Asp15:
| Fragment | Residues | Length | Localises |
|---|
| E1 | 1-3 | 3 | N-terminal Tyr1, Aib2 |
| E2 | 4-9 | 6 | Thr5, Phe6, Thr7, Ser8, Asp9 |
| E3 | 10-15 | 6 | Tyr10, Ser11, Ile12, Aib13, Leu14, Asp15 |
| E4 | 16-39 | 24 | Lys16, the acylated Lys20, Trp25, the PPP region, the C-terminal amide |
Better at the N-terminus, still bad at the C-terminus. The real answer is that no single enzyme handles this sequence, and a serious map uses two or three in parallel and stitches overlapping fragments together:
- Chymotrypsin cleaves after aromatic residues and Leu: Tyr1, Phe6, Tyr10, Leu14, Phe22, Trp25, Leu26. That is seven sites, and crucially several of them are in the C-terminal half that trypsin and Glu-C leave intact. Chymotrypsin is the enzyme that breaks up T2.
- Asp-N cleaves N-terminal to Asp, giving fragments offset by one from the Glu-C set, which is exactly what you want for overlap.
- Non-specific digestion with pepsin or a short thermolysin incubation gives a shotgun of overlapping fragments; messier to interpret, excellent for localisation.
The PPP motif near the C-terminus is genuinely hard for everything. Proline resists cleavage on its N-terminal side by most proteases and suppresses CID fragmentation, so residues 36 to 39 tend to be the least well characterised part of the molecule in any map. A report that quietly puts them inside a large fragment and calls it 100% has not lied; it has just not done the difficult part.
What to look for on a mapping report instead of coverage
- Number of fragments and the longest fragment. If the longest fragment is over about 15 residues, the map has poor localising power in that region.
- Whether MS/MS was acquired on every fragment or only on some. Accurate mass on a fragment confirms composition; MS/MS confirms sequence. A "map" with only fragment masses and no fragment spectra is a digest mass list, and it cannot distinguish a transposition or place a modification within a fragment.
- Whether more than one enzyme was used. Single-enzyme maps on peptides with sparse cleavage sites are structurally limited, and this molecule is the textbook case.
- Explicit confirmation of the modification site. The one thing you most want stated is that the acyl side chain was localised to Lys20 by MS/MS, not merely that a fragment containing Lys20 had the right total mass. Those are different claims.
- Missed-cleavage products, assigned as such. Their presence at high abundance means the digest was incomplete and the coverage claim is soft.
- Digest conditions: enzyme ratio, pH, temperature, duration. Without these you cannot evaluate any modification finding, for the artefact reasons covered elsewhere in this tag.
Your report is not fraudulent. It is a single-enzyme map on a sequence that punishes single-enzyme maps, presented with the most flattering metric in front. Ask whether they can add a chymotryptic digest, and ask for the MS/MS confirmation of the Lys20 acylation site specifically. Those two additions turn it into a real characterisation.
edited 26 Dec 2025 by pierce_count — added a caveat about sampling
8Longest-fragment length as the headline metric instead of coverage percent. That is the correction the whole field needs. – ben_akintola 9 months ago The distinction between "a fragment containing Lys20 had the right mass" and "the side chain was localised to Lys20" is the crux. Vendors conflate them constantly. – tess_amankwah 32 days ago 6Chymotrypsin plus trypsin on tirzepatide is what I have seen done properly. Two digests, one report. – h_pergande 3 months ago add a comment