Molecular Weight

The molecular mass of a peptide is the sum of atomic masses of all its constituent atoms after peptide bond formation (each amide bond involves loss of one water molecule by condensation). It is a fundamental identity parameter: two peptides of the same sequence have the same molecular mass, and any deviation from the calculated theoretical value signals a sequence error, a chemical modification, or an impurity.

Common units are the dalton (Da) and the kilodalton (kDa, 1 kDa = 1000 Da). Historically defined as 1/12 of the mass of a ¹²C atom, the recent definition (2019, new SI) sets the atomic mass constant at m_u = 1.66053906660(50) × 10⁻²⁷ kg. A 10-residue peptide typically weighs 1-1.5 kDa; a peptide hormone like insulin weighs 5.8 kDa; a monoclonal antibody is around 150 kDa.

Two mass notions coexist and must not be confused. Monoisotopic mass sums the exact masses of the most abundant isotopes of each element (¹H = 1.00783 Da, ¹²C = 12.00000, ¹⁴N = 14.00307, ¹⁶O = 15.99491, ³²S = 31.97207). It corresponds to the most intense peak in a high-resolution MS spectrum of a small molecule or short peptide. Average mass accounts for natural isotopic distribution weighted by abundance (C = 12.011, N = 14.007, etc.). It is more relevant for large proteins where the monoisotopic peak becomes undetectable in favor of a broad distribution. The difference between the two grows with mass: negligible for a dipeptide (< 0.01 Da), significant for a 50 kDa protein (several Da).

Molecular mass measurement methods are dominated by mass spectrometry. MALDI-TOF for singly charged peptides up to 20 kDa (5-50 ppm accuracy), ESI-QTOF or ESI-Orbitrap for multiply charged peptides and proteins (1-10 ppm accuracy). Simple MS mode gives the native mass; MS/MS with fragmentation (CID, HCD, ETD) confirms the sequence by analyzing b and y fragment ions. Historically, SDS-PAGE gel electrophoresis and size-exclusion chromatography estimated mass within 10-20 %, vastly insufficient accuracy for modern quality control.

Peptide identity verification on a certificate of analysis reads as follows: theoretical mass calculated from the sequence (for example 1420.62 Da monoisotopic for a given peptide), observed mass by MS (for example 1420.615 Da), ppm difference (≈ 4 ppm here), acceptance if < 10 ppm in practice. A larger difference signals a sequence error, an unintended modification (oxidation +16 Da, deamidation +1 Da, acetylation +42 Da), or a major impurity co-eluting with the product.