Counting individual sulfur atoms in a protein by ultrahighresolution Fourier transform ion cyclotron resonance mass spectrometry: Experimental resolution of isotopic fine structure in proteins

A typical molecular ion mass spectrum consists of a sum of signals from species of various possible isotopic compositions. Only the monoisotopic peak (e.g., all carbons are (12)C; all nitrogens are (14)N, etc.) has a unique elemental composition. Every other isotope peak at approximately integer multiples of ∼1 Da higher in nominal mass represents a sum of contributions from isotope combinations differing by a few mDa (e.g., two (13)C vs. two (15)N vs. one (13)C and one (15)N vs. (34)S, vs. (18)O, etc., at ∼2 Da higher in mass than the monoisotopic mass). At sufficiently high mass resolving power, each of these nominal-mass peaks resolves into its isotopic fine structure. Here, we report resolution of the isotopic fine structure of proteins up to 15.8 kDa (isotopic (13)C,(15)N doubly depleted tumor suppressor protein, p16), made possible by electrospray ionization followed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass analysis at 9.4 tesla. Further, a resolving power of m/Δm(50%) ≈8,000,000 has been achieved on bovine ubiquitin (8.6 kDa). These results represent a 10-fold increase in the highest mass at which isotopic fine structure previously had been observed. Finally, because isotopic fine structure reveals elemental composition directly, it can be used to confirm or determine molecular formula. For p16, for example, we were able to determine (5.1 ± 0.3) the correct number (five) of sulfur atoms solely from the abundance ratio of the resolved (34)S peak to the monoisotopic peak.