Sum formula annotation in high-resolution mass spectrometry using isotopic abundances?

02 February 2015 8 2K Report

I'm interested in doing sum formula annotation to highly resolved metabolites in high-resolution mass spectrometry via the isotopic peaks. A nice summary of how this is done I've found here:

http://www.sepscience.com/Information/Archive/MS-Solutions/254-/MS-Solutions-5-The-Role-of-Isotope-Peak-Intensities-Obtained-Using-Mass-Spectrometry-in-Determining-an-Elemental-Composition-Part-1

In short, the intensity of the first 13C-isotopic peak is the C-number of the monoisotopic mass times 1.1, meaning a compound with 10 12C atoms (at 100%) has a 9 12C and 1 13C peak at a relative intensity of 11%, the so-called X+1-peak. The second isotopic peak, X+2, is calculated by 0.006 × nC2.

Now, I'm having trouble with a certain metabolite with the sum formula C55H57O29 (see attached file). At this oxygen count, I assume the 18O peak already can have an impact at the isotopic peaks. The according isotopic peak can be calculated by 0.20 times nO. I'm unsure of the composition of the second isotopic peak (m/z 1183) of my compound. The first isotopic peak, m/z 1182 has one 13C-Atom and an intensity of 55 x 1.1, which correctly is 60.5, as can be seen. m/z 1183, purely considering 13C, and not 18O, should have an intensity of 55² x 0.06 = 18.15, but I see an intensity of 23.97. However, the annoted sum formula from purely 13C is very good (0.48 ppm). If I consider now that m/z 1183 also contains one 18O-Atom, 0.20 x 29 = 5.8. Because isotopic peak abundances are additive for each element, this would be 18.15 + 5.8 = 23.95, which fits near perfectly for the intensity I actually see (23.97)

However, the annotated sum formula with two 13C and one 18O, C5313C2H57O2818O, has a mass of 1185 m/z, and I actually see this as a very good first sum formula offer at 0.66 ppm. Did I make some mistake in the calculations of m/z 1183, or how can it be that the intensity is 23.97, while it should be 18.15, assuming 18O doesn't factor in already at the second isotopic peak - as the first isotopic peak where it occurs is obviously m/z 1185?

Thanks in advance.

David P A Kilgour

Hi,

The approach you are using for generating an isotopic model only really works for very small molecules with few heteroatoms. For your purposes, you might find that this website helps you with performing the calculations - http://www.sisweb.com/mstools/isotope.htm

For more in depth reading, try - Rockwood, A. and Palmblad, M.: Isotopic Distributions. in Mass Spectrometry Data Analysis in Proteomics, vol. 1007: Methods in Molecular Biology (R. Matthiesen ed; Humana Press, p. 65-99 (2013)

Or - Kubinyi, H.: Calculation of isotope distributions in mass spectrometry. A trivial solution for a non-trivial problem. Anal. Chim. Acta 247, 107-119 (1991)

Good luck,

David

Ali Farhat

yes as David said, larger molecules are more difficult to work with using this model. It applies more for the smaller molecules.

Robert Cody

Here are the theoretical isotope abundances (ignoring the electron mass for the exact m/z):

m/z Rel. Abundance

1181.29855 100.0000 **************************************************

1182.30195 61.2470 *******************************

1183.30473 24.3930 *************

1184.30748 7.2820 ****

1185.31011 1.7960 *

1186.31271 0.3810

1187.31526 0.0720

1188.31778 0.0120

1189.32029 0.0020

Alan Rockwood

If you want to calculate the isotopic composition of the isotopic peaks (e.g. how much 12C, 13C, 1H, 2H, 16O, 17O, 18O, etc. there is in each nominal isotopic peak) then you might find the following reference useful.

Isotopic compositions and accurate masses of single isotopic peaks. Journal of the American Society for Mass Spectrometry, Volume 15, Issue 1, January 2004, Pages 12–21.

Alan Rockwood

Also, if you want a program that is probably the most efficient one for calculating accurate abundances and accurate masses consider emass, which is described in the reference

Efficient Calculation of Accurate Masses of Isotopic Peaks. Journal of the American Society for Mass Spectrometry, Volume 17, Issue 3, March 2006, Pages 415–419.

There is a website where you can download the emass program, or you could contact me for a copy.

Hannes Doerfler

Thanks a lot for your answers Mr. Cody and Prof. Rockwood, they are very helpful to me! I also downloaded the publications and emass; I already tested it and it will be very useful for my work!

Thanks again and best regards.

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