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

Abstract

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 12C; all nitrogens are 14N, etc.) has a unique elemental composition. Every other isotope peak at approximately integer multiples of approximately 1 Da higher in nominal mass represents a sum of contributions from isotope combinations differing by a few mDa (e.g., two 13C vs. two 15N vs. one 13C and one 15N vs. 34S, vs. 18O, etc., at approximately 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 13C,15N 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/Deltam50% approximately 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 34S peak to the monoisotopic peak.

Figure 1

ESI FT-ICR mass spectrum (Upper Left), from a single time-domain data acquisition, of bovine insulin. Theoretical (Upper Right) and experimental (Lower) isotopic fine structure is shown for the isotopic peak (★) ∼5 Da above the monoisotopic mass. Individual elemental compositions are clearly resolved at approximately correct relative abundances.

Figure 2

Theoretical (Left) and experimental (Right) mass spectra of bovine ubiquitin. The isotopic distribution (m/Δm_50% ≈ 100,000) is shown above the dotted line, and the isotopic fine structure (m/Δm_50% ≈8,000,000) for each of several isotopic peaks (at ≈2, 3, 4, 5, and 6 Da above the monoisotopic mass) appears below the dotted line. The experimental data were obtained from a single time-domain data set for ions of the isolated 9+ charge state. Note the good match between experimental and theoretical fine structure.

Figure 3

Mass scale-expanded spectra of one isotopic peak for ¹³C,¹⁵N doubly depleted p16 tumor suppressor protein, from a single ESI FT-ICR mass spectrum. The theoretical isotopic distribution is shown at upper left, in which ★ denotes the isotopic peak ≈2 Da above the monoisotopic mass. The theoretical isotopic fine structure for the starred peak is shown at upper right. Double-depletion improves the SNR of the monoisotopic, ³⁴S, and ¹⁸O species, by reducing the abundances of species containing ¹³C and/or ¹⁵N.

Figure 4

Relative abundance (ordinate) and mass difference (abscissa) resulting from substitution of a single heavy isotope for the most abundant (lower-mass) isotope of that element. (The abundance for deuterium is magnified by a factor of 10 to make it more visible.) For ease in computation, each mass difference has been adjusted for the nominal mass difference between the two isotopes. Each displayed mass difference is thus like a mass defect (the difference between exact and nominal mass), in the sense that the mass shift is a small fraction of 1 Da. These values facilitate the identification (by abundance and accurate mass) of various fine structure components at a given nominal mass (see text).

Figure 5

Mass scale-expanded spectra of three isotopic peaks of bovine insulin, obtained by coadding 11 frequency-domain ESI FT-ICR spectra. The theoretical isotopic distribution is shown at upper left, in which ★ denote the monisotopic peak (Left), and isotopic peaks ∼2 Da (middle) and ∼4 Da (Right) above the monoisotopic mass. Note the significant improvement in SNR (compare with Fig. 1) because of frequency-domain signal averaging.

Figure 6

Mass-scale expanded spectra of two isotopic peaks of ¹³C, ¹⁵N doubly depleted p16 tumor suppressor protein, obtained by coadding nine frequency-domain ESI FT-ICR spectra. The theoretical isotopic distribution is shown at upper right, in which ★ denote the monisotopic peak (Left), and isotopic peak (Right) ∼2 Da above the monoisotopic mass. From the abundance ratio of the (resolved) ³⁴S to the monoisotopic species, we determined the number of sulfur atoms in the protein to be 5.1 ± 0.3 without use of any other information about the protein.