Quantification

To quantify signals, normally an internal standard is used to compare the signal intensity with the intensity of the standard. In mass spectrometric analysis, it is crucial to use a standard with similar physico-chemical properties, as ionisation should take place with a similar efficiency. Furthermore, the standard must neither interact with the sample (e.g. by forming adducts) nor inhibit the ionisation process. Moreover, monitoring an enzymatic reaction, the standard must not interact with the enzyme or disturb the enzymatic reaction. As it is almost impossible to find a standard which meets all these requirements, a different approach was undertaken; due to the known stoichiometry of the enzymatic reaction, the concentration of each compound involved in the reaction depends on each other and the possible concentration ratios (or relative concentrations) can be determined easily (see below). Thus correlating the relative signal intensities (see below) and the relative concentrations allows the quantification of the reactants without an internal standard.

The possible relative concentrations of each reactant can be obtained as described in the following equations ($n_x$: number of molecules of compound $x$):

$$\left[n_{GpC} - n \right] \longrightarrow \left[(n_{Cyt} + n) + (n_{cGMP} + n)\right]$$

$$\left[n_{cGMP} -n\right] \longrightarrow \left[(n_{GMP} + n)\right]$$

Figure 3.1 shows the possible relative concentrations for the compounds involved in the enzymatic cleavage of 5',3'-GpC by RNase T1. Comparing the relative intensities ($i$) with the relative concentrations ($c$) (see equations 3.1 and 3.2, $I$: absolute intensity, $C$ absolute concentration)

$$i_n = \frac{I_n}{\sum I}$$ (3.1)

$$c_n = \frac{C_n}{\sum C}$$ (3.2)

allows the development of calibration curves to estimate the relative concentration of each compound in the sample. As the initial concentration of the substrate is known, the concentration for each compound can be calculated from the known relative concentration.

Figure 3.1: Relative concentrations (c) of the compounds involved in the enzymatic cleavage of 5',3'-GpC by RNase T1. The red line represents the relative 5',3'-GpC concentration, the green line the relative cytidine concentration. The blue area represents the possible relative concentration range for 2',3'-cGMP and 3'-GMP.

Figure 3.2: Relative concentrations (c) and relative intensities (i) for 5',3'-GpC. The red line represents the first branch (0$<i<$0.22) of the calibration curve (r$^2=$0.89), the green line the the second branch (r$^2=$0.90; 0.22$\le i\le$1).
Conditions: Initial 5',3'-GpC concentration (c=1): 1,000$\mu$M, eluent: methanol:water:acetic acid 66:33:1, positive ion mode, instrument parameters as described in 2.2.2.1

Figure 3.2 shows the calibration curve for 5',3'-GpC acquired under the same conditions as the reaction data. The data were fitted using a MARQUARDT-LEVENBERG nonlinear least-square fitting algorithm to two calibration curves (see equation 3.3), as the data show a kink at approximately $i=$ 0.22. The correlation coefficients ($r^2$) are 0.89 for the first branch (0 $<i<$ 0.22) and 0.90 for the second (0.22 $\le i\le$ 1). The calibration curve for the quantification derived from the data acquired is:

$$c_{GpC}(i) = \begin{cases}0.63865 \times i - 0.033108 & 0 < i < 0.22 \\ 1.29146 \times i - 0.180183 & 0.22 \le i \le 1 \end{cases}$$ (3.3)

The quantification of 2',3'-cGMP was performed in the same way, using equation 3.4 ($r^2 =$ 0.91) as calibration curve.

$$c_{cGMP}(i) = 1.17709 \times i - 0.299923$$ (3.4)