Expected Mass Table for Peptide Mass Spectrometry: Average vs Monoisotopic, Theoretical vs Observed

A mass spectrometry result on an analytical report compares an observed mass with an expected (theoretical) mass. Whether the two "match" depends on which theoretical mass is being used — average or monoisotopic — and on how the instrument reports the ion. This page explains the terms and tabulates expected values for the compounds listed in the catalog, computed from their recorded molecular formulas.

Average vs Monoisotopic Mass

Every element occurs as a mixture of isotopes. The monoisotopic mass is the mass of the molecule built entirely from the most abundant isotope of each element (¹²C, ¹H, ¹⁴N, ¹⁶O, ³²S). The average mass uses the natural-abundance-weighted atomic weights (C 12.011, H 1.008, N 14.007, O 15.999, S 32.06) and is the "molecular weight" printed on most product pages and reports. For a peptide of a few hundred daltons the two differ by well under a dalton; for a 4–5 kDa peptide the average mass is roughly 2–3 Da higher than the monoisotopic mass, because the probability of containing one or more ¹³C atoms rises with the number of carbons.

Which one an instrument reports depends on resolution. Low-resolution ESI or MALDI-TOF instruments see the isotope cluster as a single unresolved peak whose centroid corresponds to the average mass. High-resolution instruments resolve the individual isotope peaks; software then reports either the monoisotopic peak or a deconvoluted average, and the report should state which.

Theoretical vs Observed

The theoretical mass is calculated from the molecular formula. The observed mass is measured. Typical agreement is within ±0.1% for low-resolution ESI (about ±1.4 Da at 1,420 Da and ±5 Da at 5,000 Da) and within a few parts per million on high-resolution instruments. A discrepancy larger than the instrument tolerance means the ion measured is not the expected molecule: a deletion sequence (−57 Da for a missing glycine, −71 Da for alanine, and so on), an oxidized methionine (+16 Da), a deamidated asparagine or glutamine (+1 Da), a retained protecting group, or a different compound altogether.

Charge States and m/z

Electrospray ionization produces multiply protonated ions, and a mass spectrometer measures the mass-to-charge ratio (m/z) rather than the mass. For an ion carrying z protons, m/z = (M + z × 1.00728) / z, where 1.00728 Da is the proton mass. Peptides with several basic residues commonly appear as [M+2H]²⁺ and [M+3H]³⁺ in ESI; the deconvoluted mass on the report has already been converted back to M. Sodium adducts (+21.98 Da relative to the protonated ion) and potassium adducts (+37.96 Da) also occur and are not sequence errors.

Expected Mass Table

Values below are computed from the molecular formula of the free (neutral) peptide as recorded for each listed compound; the catalog molecular weight is shown alongside for comparison. Approximate m/z values use the computed average mass, or the catalog value where no formula is recorded. Counter-ions (TFA, acetate) are not part of the ion mass — see TFA vs acetate salts. Compounds without a parseable formula show a dash in the computed columns.

Computed columns use IUPAC standard atomic weights for the average mass and the principal-isotope masses for the monoisotopic mass. A difference between the computed average mass and the catalog molecular weight usually reflects rounding, a different set of atomic weights, or a catalog value that refers to a salt or complex rather than the free peptide.

Reading the MS Line on a Report

Look for three things: the expected value and whether it is stated as average or monoisotopic; the observed value and the instrument type (ESI, MALDI-TOF, or high-resolution); and the difference, ideally expressed both in daltons and as a percentage or in ppm. The analytical reports (COA) that Pepta Labs makes available by email request include a mass spectrometry identity check performed by an independent laboratory commissioned through a cooperative purchasing group; the report may name the group or source rather than Pepta Labs, and Pepta Labs does not test in-house. The reported HPLC purity is shown on each product page. For how purity and identity are measured, see HPLC vs mass spectrometry.