Peptide identity testing with mass spectrometry

Purity tells you how clean a sample is. Identity tells you whether it's the right molecule. For synthetic peptides, identity is usually confirmed by mass spectrometry (MS).

What MS measures

Mass spectrometry measures the mass of the molecules in a sample. The lab compares the observed mass with the theoretical mass calculated from the peptide's sequence. A match within the instrument's accuracy supports identity [1].

Two common methods

• MALDI-TOF: a fast check of the intact mass, well suited to routine identity confirmation [1].
• LC-MS (liquid chromatography with electrospray MS): separates the sample first, then measures each component. Because peptides pick up multiple charges, the software converts the readings back to a single neutral mass. LC-MS can tie a mass to a specific HPLC peak, which helps identify impurities [1].

Extra peaks you may see

• +16 Da: an oxidized form, often at methionine [1].
• Sodium or potassium adducts: the same molecule carrying a sodium or potassium ion [1].
• Lower masses: deletion or truncated sequences [1].

What intact mass can't prove

• Purity: that comes from HPLC peak areas, not MS [1].
• Sequence order: two peptides with the same amino acids in a different order have the same mass. Sequence-level confirmation needs fragmentation (MS/MS) [1].
• Amount: MS identity doesn't measure how much peptide is in the vial; that's net peptide content.

What to look for on a report

An identity result should show the observed mass next to the expected mass, name the method, and come from the same lot as the purity result. See how to read a peptide COA.

Questions

How do you confirm a peptide's identity?

Usually by mass spectrometry, comparing the observed molecular weight with the theoretical weight from the sequence. A match supports identity.

What is the difference between MALDI-TOF and LC-MS?

MALDI-TOF is a quick intact-mass check. LC-MS separates the sample first and measures each component, so a mass can be matched to a specific HPLC peak.

Can mass spectrometry prove the sequence?

Intact mass alone can't, because sequences with the same amino acids in a different order have the same mass. Fragmentation (MS/MS) gives sequence-level evidence.