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Browse CatalogMass spectrometry (MS) is one of the essential analytical techniques in peptide science. While HPLC tells you how pure your peptide is, mass spectrometry tells you what your peptide actually is. It does this by measuring the molecular mass of the compound with extreme precision, allowing you to confirm that the synthesis produced the correct sequence. Every batch of NXPeptides product undergoes MS analysis as part of our quality assurance process.
A mass spectrometer works by converting molecules into ions (charged particles), separating those ions based on their mass-to-charge ratio (m/z), and detecting them. The result is a mass spectrum: a plot of signal intensity versus m/z. Each peak in the spectrum corresponds to an ion with a specific mass.
For peptides, the most important peak is the one corresponding to the intact molecular ion, since its mass directly confirms the identity of the peptide. If the observed mass matches the theoretical mass calculated from the amino acid sequence, you can be confident that the correct peptide was synthesized.
ESI is the most commonly used ionization method for peptide mass spectrometry. It works by spraying the peptide solution through a charged capillary, creating a fine mist of charged droplets. As the solvent evaporates, multiply charged peptide ions are produced.
A key feature of ESI is that it produces multiply charged ions. A peptide might pick up 2, 3, or even more protons, resulting in ions with charge states of +2, +3, etc. This means you will see multiple peaks in the spectrum for the same peptide, each corresponding to a different charge state:
This charge state distribution is actually useful because it provides multiple independent measurements of the molecular weight, increasing confidence in the identification.
MALDI is the other major ionization technique used for peptides. In MALDI, the peptide is mixed with a UV-absorbing matrix compound and spotted onto a metal plate. A laser pulse hits the spot, causing the matrix to absorb energy and transfer it to the peptide, launching it into the gas phase as an ion.
Unlike ESI, MALDI typically produces singly charged ions [M+H]+, which makes the spectrum simpler to interpret. MALDI is particularly good for:
When you look at the mass spectrum on your Certificate of Analysis, here is what to pay attention to:
The dominant peak(s): These should correspond to your peptide. For ESI, you may see a series of multiply charged peaks. For MALDI, you should see one major peak at [M+H]+.
Observed vs. Expected mass: The COA will list both values. For a positive identification, the observed mass should match the expected mass within the instrument’s accuracy. For modern instruments, this is typically within 0.01% or better.
What counts as a match? For peptides under 3000 Da, observed mass should be within 1 Dalton of the expected value. For larger peptides, within 0.05% relative error is considered a good match. Slight deviations (0.5 to 1 Da) can result from sodium or potassium adducts, TFA counter-ions, or isotope peaks being selected instead of the monoisotopic peak.
HPLC purity alone is not sufficient for full quality assurance. A peptide could be very pure by HPLC (one clean peak) but could be the wrong peptide entirely if a synthesis error went undetected. Mass spectrometry catches these errors. It confirms that the molecular weight matches the expected sequence, giving you confidence that you are working with the right compound.
This is especially important for:
In many quality control workflows, HPLC and MS are combined in a single instrument called LC-MS (Liquid Chromatography-Mass Spectrometry). The HPLC separation feeds directly into the mass spectrometer, so each peak in the chromatogram also gets a mass measurement. This is an extremely powerful approach because it tells you both the purity AND the identity of every component in the mixture.
At NXPeptides, we use LC-MS as part of our comprehensive quality control process. Learn more about our testing protocols on our Quality Assurance page.
If you have questions about the mass spec data on your COA or need help interpreting results, email us at support@nxpeptides.com or visit our Contact page.
All products sold by NXPeptides are strictly for research use only and are not intended for human consumption.