Mass spec for identity confirmation: a quick primer

If HPLC answers “how pure?”, mass spec answers “is this the right molecule?” Here’s how to read the MS section of a COA.

The basics

• Most peptide COAs use ESI-MS (electrospray) or MALDI-TOF.
• ESI usually produces multiply charged ions, so you’ll see a series of peaks at m/z = (M + nH)/n. The software deconvolutes these back to a single molecular mass.
• MALDI typically shows mostly singly charged [M+H]+ ions, which are easier to read by eye.

What to check

• Observed vs. theoretical mass. The COA should list both. For small peptides, agreement within about 1 Da is typical for routine instruments, and high-resolution instruments should be much tighter.
• Monoisotopic vs. average mass. Make sure you’re comparing like with like. The two can differ by more than 1 Da for larger peptides.
• Common adducts. Peaks at +22 (Na) or +38 (K) relative to [M+H]+ are usually adducts, not impurities.
• Telltale modifications. +16 often means oxidation (for example, Met), and +1 can indicate deamidation. Masses well below the target can point to truncated sequences.

Limits

Standard MS confirms mass, not sequence. Two peptides with the same composition in a different order can have identical masses, and so can D/L isomers. Sequence confirmation needs MS/MS fragmentation. For most routine verification, though, a matching mass plus a good HPLC trace is a reasonable baseline.

Post spectra below if you want help interpreting one.