Peptide Mapping¶

Sequence Confirmation by HPLC-MS/MS¶

Purpose¶

Peptide mapping confirms the primary sequence and detects sequence variants, post-translational modifications, and degradation products. It is the most definitive identity test for peptide therapeutics.

Workflow¶

flowchart LR
    A[Sample] --> B[Reduce & Alkylate];
    B --> C[Proteolytic Digestion];
    C --> D[HPLC Separation];
    D --> E[MS/MS Fragmentation];
    E --> F[Sequence Assignment];
    F --> G[Coverage Report];

Proteolytic Enzymes¶

Enzyme	Cleavage Site	Avg Fragment Length	Application
Trypsin	C-term of Lys, Arg	5–15 AA	Standard mapping
Glu-C (V8)	C-term of Glu, Asp	8–20 AA	Hydrophobic regions
Lys-C	C-term of Lys	10–25 AA	Long peptides
Chymotrypsin	C-term of Phe, Tyr, Trp	4–12 AA	Complementary coverage
Pepsin	Broad specificity	3–10 AA	Acidic conditions

Digestion Protocol (Trypsin)¶

Step	Condition	Duration
Denature	6 M guanidine-HCl, pH 8.0	30 min
Reduce	10 mM DTT, 56 °C	30 min
Alkylate	25 mM iodoacetamide, RT, dark	30 min
Buffer exchange	50 mM ammonium bicarbonate	—
Digestion	Trypsin 1:50 (w/w), 37 °C	4–16 h
Quench	0.1% TFA final	—

Sequence Coverage Targets¶

Application	Target Coverage
Identity confirmation	≥80% sequence coverage
Variant detection	≥95% sequence coverage
Biosimilar comparability	≥95% + 100% disulfide mapping
Release testing	Matches reference map

Data Interpretation¶

MS1: Identify peptide mass → match predicted digest
MS/MS (CID/HCD): b/y ion series → confirm sequence
Coverage: % of amino acids confirmed by MS/MS
Modifications: +57 Da (carbamidomethyl Cys), +16 Da (Met oxidation)

🔗 Related: LC-MS Testing | Mass Confirmation | Peptide Quality Control Guide