Mass Spectrometry Explained: How Peptide Identity Is Confirmed

The peptide is first converted into gas phase ions. Electrospray ionization (ESI) sprays a solution through a charged needle and typically produces ions carrying several charges, while matrix assisted laser desorption ionization (MALDI) uses a laser and a light absorbing matrix and mostly produces singly charged ions. The analyzer then separates ions by mass to charge ratio, and software converts the pattern back to a molecular mass.

When the instrument is coupled to liquid chromatography (LC-MS), each chromatographic peak can be assigned a mass. That pairing is the strongest routine way to show both what the main component is and what the minor peaks are.

The expected value comes from the amino acid sequence plus any modifications such as C terminal amidation, acetylation, cyclization or a metal complex. Reports often list monoisotopic or average mass; check which one is being compared. Small differences within the stated tolerance are normal. A difference near 16 Da can indicate oxidation, near 18 Da a water loss or gain, and differences matching one residue can indicate a deletion or truncation.

Tandem MS (MS/MS) breaks selected ions into fragments and reads the fragment ladder. That gives sequence level evidence and can locate where a modification sits, which an intact mass alone cannot do.

• Isomers with the same mass, including some D and L amino acid swaps, can look identical by intact mass.
• Ionization efficiency differs between molecules, so peak heights in a mass spectrum are not a purity measurement.
• A single spectrum from a different lot says nothing about the lot in hand.