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Browse CatalogNovember 19, 2025
Mass spectrometry (MS) is an indispensable analytical technique for confirming the molecular identity of synthetic peptides. While HPLC measures purity, it cannot definitively verify that the major peak corresponds to the correct peptide. Mass spectrometry fills this critical gap by measuring the mass-to-charge ratio (m/z) of ionized peptide molecules, allowing researchers to confirm that the observed molecular weight matches the theoretical molecular weight calculated from the amino acid sequence. Common ionization methods for peptides include electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).
Electrospray ionization mass spectrometry (ESI-MS) generates multiply charged ions, producing a characteristic envelope of peaks that can be deconvoluted to determine the intact molecular mass. This method is well-suited for peptides ranging from small fragments to larger sequences of 50 or more amino acids. MALDI-TOF (Time of Flight), by contrast, typically produces singly charged ions and offers rapid, high-throughput analysis with minimal sample preparation. Both techniques provide mass accuracy sufficient to detect single amino acid substitutions, deletions, or additions in the peptide chain.
When reviewing MS data on a Certificate of Analysis, researchers should compare the observed mass with the expected monoisotopic or average molecular weight, accounting for the counterion form (e.g., acetate or trifluoroacetate salt). A mass deviation of less than 0.1% from the theoretical value generally confirms correct identity. Discrepancies may indicate incomplete deprotection during synthesis, unexpected modifications such as oxidation of methionine residues, or the presence of adducts. Tandem mass spectrometry (MS/MS) can provide additional sequence-level confirmation by fragmenting the peptide and analyzing the resulting ion series.