Table 1.
Method Comparison Summary.
Figure 1.
Different methods used for fitting a single peptide isotopic cluster to a MALDI-TOF spectrum of unlabeled Angiotensin II.
The inclusion of a slope-intercept form baseline (red estimation) increases the fit over a flat baseline (blue estimation). Both of which are better than not including a baseline (green estimation).
Figure 2.
A MALDI-TOF mass spectrum from the analysis of Ang I extracellular breakdown [2] by rat glomeruli in the presence of amastatin (APA inhibitor) and thiorphan (NEP inhibitor) at 60 minutes.
The sample contains a mixture of Ang-(2–10), Ang-(1–9), and SIS-Ang-(2–10) that overlap forming one cluster. These peaks are fit and the individual areas for each isotopic cluster can be decomposed from the spectrum.
Figure 3.
A MALDI-TOF mass spectrum of a known ratio of 1∶1∶1∶1 peptides consisting of 300 nM Ang-(2–10), Ang-(1–9), SIS-Ang-(2–10) and SIS-Ang-(1–9).
The spectrum has been fit using GMM and the figure shows how each estimated peptides contributes to the whole spectrum. Since all peptides are estimated simultaneously, each peptide is presented here separately to illustrate the individual contribution of each peptide to the spectrum as a whole. (A) Figure 3a shows the entire estimation as a whole, preformed as a single fit to a single cluster of four overlapping peptides. The data is shown in black with the estimated peaks superimposed in red. (B) Figure 3b shows the estimated contribution of Ang-(2–10) to the spectra superimposed in blue. (C) Figure 3c shows the estimated contribution of Ang-(1–9) to the spectra superimposed in green. (D) Figure 3d shows the estimated contribution of SIS-Ang-(2–10) to the spectra superimposed in dark yellow. (E) Figure 3e shows the estimated contribution of SIS-Ang-(1–9) to the spectra superimposed in dark purple.
Figure 4.
Correlation plots showing the difference in estimation error of the peak ratio for a given spectrum when different methods of peak ratio measurement.
The red line denotes a correlation of ρ = 1 and the blue lines denote 0% error in ratio estimation for that given method. Here we see that the Peak intensity and Riemann sum AUC methods of quantification correlate more highly with one another than with the Gaussian mixture method. Note that the GMM estimates tend to cluster closer to the blue line suggesting lower error.
Table 2.
Two way ANOVA with pairwise testing.
Table 3.
Secondary peptide ratio estimation.