March 6, 2015

Tutorial: Mass spectrometry in plant science, part 2 – using isotopic labels to study plant metabolism



In this second entry on the use of mass spec in plant research, I want to introduce isotopic labeling and its power to inform us about plant metabolic processes quantitatively. By feeding an isotopically labeled substrate into the metabolic stream of an organism and observing it later in some downstream metabolic intermediate, we can gain insights into the rates at which these transformations take place inside the cell. Experimentally, we observe these isotopic labels as shifts to heavier m/z values in the mass spectrum of molecules that incorporate them. As isotopically labeled atoms introduced in our labeling experiment migrate through the different metabolites in a biochemical pathway, we see specific mass peaks rise and fall in a spectrum over time that are indicative of the turnover of that pool of metabolites. This is a complex subject that has been covered in detail with precisely the mathematical rigor you might expect (see for example publications by Wolfgang Wiechert on metabolic flux analysis). In this short monograph, I can only hope present how well mass spectral analysis lends itself to quantifying plant metabolism.


Let me begin with the definition of nominal atomic mass, which is the mass of a molecule when all its atoms are represented by the lightest and most abundant isotope of an element (at least on earth). It is useful to contrast this with the definition of average molecular weight, which is the averaged weight of an element or compound taking natural isotopic abundance into consideration. So even though the nominal mass of chlorine gas (35Cl2) is 34.9689 x 2 = 69.9378, its average molecular weight is 35.453 x 2 = 70.906 due to the high abundance of 37Cl, which is about 25% of all Cl on earth. The remaining 75% is the 35Cl isotope, and this is the isotope we use to calculate its nominal mass. Reviewing how a quadrupole works in part 1, it should be obvious that Cl2 produces a mass spectrum with a base peak at m/z 70 (at unit resolution) and smaller peaks at m/z 72 and 74 (the M+2 and M+4 peaks caused by one or two 37Cl atoms, respectively). No peak is seen at m/z 71 since this represents an averaged weight taking into account all the molecules in a sample at once, whereas the mass spectrometer measures the abundance of ions at specific masses individually, so a given pair of Cl atoms can only produce a combined weight of m/z 70, 72, or 74.
In biochemistry, the nominal masses of elements we need to worry about are correspond to 1H, 12C, 14N, 16O, 31P, and 32S. Silicon and the halogens rarely make an appearance in biology but may be useful in the derivitization of some metabolites to make them more amenable to analysis. The heavier isotopes of the socalled SPONCH elements are present in terrestrial material in essentially fixed amounts, but they are usually only a small percentage of the lightest isotope. In the table below, we can see the isotopic abundances of the elements with significance in biology.


Isotope
Mass
% abundance
1H
1.0078
99.99
2H
2.0141
0.015



12C
12.0000
98.89
13C
13.0034
1.11



14N
14.003
99.63
15N
15.000
0.37



16O
15.9949
99.76
17O
16.9991
0.04
18O
17.9992
0.20



31P
30.9738
100



32S
31.9721
95.02
33S
32.9715
0.75
34S
33.9678
4.21


In mass spec, we are only interested in stable isotopes, so none of the radioactive isotopes of those elements are included. The presence of these natural isotopes complicates our ability to quantify isotopic labeling experiments since they will appear as heavier ions in the mass spectrum (i.e., X+1, X+2, etc.) just like the ions we purposefully label during our experiment. But we can compensate the raw data using the values above when the identity of the analyte is known. And even when it is unknown, we can use unlabeled control samples to get an idea of the background produced by natural isotopic abundance in the X+1, X+2, etc. mass peaks. That means that by measuring this background and making the appropriate correction, we can administer labeled substrates during labeling experiments and trace the movement of those atoms into different metabolites by mass spec with high accuracy.

For the most part, 13C is the most useful isotope in experiments of this sort. Plants have the advantage that they take up CO2 from their surroundings as part of their natural metabolism, and so 13CO2 represents a convenient manner of introducing isotopic label into the metabolic pools of plant cells without disturbing their natural metabolism (but primary isotope effects could also influence certain reactions). This turns out to be quite important since the rates of metabolic transformations we hope to measure during a labeling experiment are quite sensitive to the state of the plant. If we have to disturb the plant significantly in order to get the label inside, we would no longer be measuring the process we are interested in, or worse we may obtain a number representing a perturbed, irreproducible state and assume incorrectly it is representative of the plant’s natural metabolic rates. Another advantage of using 13CO2 as a labeling substrate is that since it contains only a single C atom, it allows us to consider label incorporation as single events that correspond to individual 13CO2 uptake events. This means that later on when we consider complex metabolites containing multiple C atoms, or pathways where the number of C atoms among metabolites changes, we can always refer back to the number of individual 13C atoms present in a molecule and draw a one-to-one correspondence to the M+1, M+2, etc. labeling series in the mass spectrum with the incorporation of 1, 2, or more molecules of 13CO2. When we finally calculate the % labeling of any metabolite pool, we can express it in terms of the fraction of C atom positions in the entire metabolite pool that are occupied by a 13C atom. Our calculated rates then come from observing how the fraction of a metabolite pool bearing a label evolves over a time course. In the next entry on mass spectrometry in plant research, I will show some specific examples of plant metabolites and their mass spectra following different periods of labeling with 13CO2 and we will see how this mass spectral data can be used to calculate % label incorporation.

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