What is peptide mapping? Enzymatic digestion and LC-MS for longer peptides

Peptide mapping cuts a long peptide or protein into predictable fragments with a protease such as trypsin, separates them by LC-MS and matches each fragment's mass to the expected sequence. The share of the sequence covered by matched fragments is the sequence coverage. It is the standard identity test for molecules too large to sequence whole.

When is peptide mapping used instead of a single mass?

For a short peptide, an intact mass plus MS/MS usually confirms identity. For longer peptides (roughly 30+ residues) and small proteins, intact MS/MS fragments poorly and isomeric changes are easy to miss; digesting first produces short pieces that fragment well.

Which enzymes cut where?

Enzyme	Cleaves after	Notes
Trypsin	Lys, Arg (not before Pro)	Most common; gives fragments of useful size
Glu-C	Glu (and Asp in phosphate buffer)	Complements trypsin for Lys/Arg-rich sequences
Chymotrypsin	Phe, Tyr, Trp (Leu, Met slower)	Useful for hydrophobic regions
Lys-C	Lys	Robust in denaturing conditions
Asp-N	Before Asp	Specific, fewer fragments

What does a peptide map show?

Sequence coverage: percentage of residues in matched fragments; high coverage supports identity.
Modifications: oxidation, deamidation or truncation localised to one fragment.
Disulfide connectivity, if the digest is run with and without reduction.
Process improvements such as automated online digestion have made mapping faster and more reproducible.

Source: Fully unattended online protein digestion and LC-MS peptide mapping, Anal Chem 2023 (PubMed 37816151); Efficient LC-MS peptide mapping using accelerated sample preparation, J Chromatogr B 2020 (PubMed 31881514)

What are its limits?

Missed cleavages and non-specific cuts complicate assignment.
Very short or very hydrophilic fragments may be lost at the solvent front.
Mapping confirms identity, not purity.

How does Sable document lots?

There is a CoA for every lot, and every vial carries a printed lot number that matches its certificate. A CoA is emailed within 2 hours of a request.

Frequently asked questions