The measurement
The sample is ionised and the ions separated by mass-to-charge ratio. The measured mass is compared against the value calculated from the sequence; agreement within instrument tolerance confirms identity.
Peptides commonly carry multiple charges, so the raw spectrum shows a series of peaks that are computationally converted back to a single molecular mass.
What mass shifts mean
Differences from the expected mass are diagnostic. Plus 16 daltons suggests oxidation. Plus 1 suggests deamidation. A deficit matching a residue mass suggests a deletion sequence. A shift matching water suggests incomplete drying or a hydrolysis product.
Reading the shift often identifies the problem without further work.
Limits
Mass spectrometry does not distinguish species of identical mass — it cannot tell leucine from isoleucine, and it will not separate sequence isomers. Sequence confirmation requires fragmentation, which is a further experiment.
It also says nothing about how much peptide is in the vial. That is a separate measurement.
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