Abstract
Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) coupled with stable isotope standards (SIS) has been used to quantify native peptides. This peptide quantification by MALDI-TOF approach has difficulties quantifying samples containing peptides with ion currents in overlapping spectra. In these overlapping spectra the currents sum together, which modify the peak heights and make normal SIS estimation problematic. An approach using Gaussian mixtures based on known physical constants to model the isotopic cluster of a known compound is proposed here. The characteristics of this approach are examined for single and overlapping compounds. The approach is compared to two commonly used SIS quantification methods for single compound, namely Peak Intensity method and Riemann sum area under the curve (AUC) method. For studying the characteristics of the Gaussian mixture method, Angiotensin II, Angiotensin-2-10, and Angiotenisn-1-9 and their associated SIS peptides were used. The findings suggest, Gaussian mixture method has similar characteristics as the two methods compared for estimating the quantity of isolated isotopic clusters for single compounds. All three methods were tested using MALDI-TOF mass spectra collected for peptides of the renin-angiotensin system. The Gaussian mixture method accurately estimated the native to labeled ratio of several isolated angiotensin peptides (5.2% error in ratio estimation) with similar estimation errors to those calculated using peak intensity and Riemann sum AUC methods (5.9% and 7.7%, respectively). For overlapping angiotensin peptides, (where the other two methods are not applicable) the estimation error of the Gaussian mixture was 6.8%, which is within the acceptable range. In summary, for single compounds the Gaussian mixture method is equivalent or marginally superior compared to the existing methods of peptide quantification and is capable of quantifying overlapping (convolved) peptides within the acceptable margin of error.
Highlights
MALDI-TOF is a convenient tool for determining peptide abundance in high-throughput workflows
Single Peak Analysis As a proof of principle, the Peak Intensity and Riemann sum area under the curve (AUC) methods of signal measure and the Gaussian mixture method were used to examine 26 spectra (9 Ang-(2–10)/stable isotope standards (SIS)-Ang(2–10) and 17 Ang-(1–9)/SIS-Ang-(1–9)) that consisted of replicate MALDI-TOF analysis of seven different mixtures
These measures were used to back calculate a ratio of native to labeled peptide which was compared to the true ratio
Summary
MALDI-TOF is a convenient tool for determining peptide abundance in high-throughput workflows. MALDI-TOF MS is a t a solid-state ionization technique in which the sample is mixed with a chemical (matrix) that is excited by an ultraviolet or infrared laser. Principles underlying each ionization technique have been well described elsewhere [1] Visualizing specific peptides or other analytes by exact mass allows for a greater degree of specificity in quantification and identification. MALDI-TOF has been used to measure angiotensin (Ang) peptides in cell culture or tissue samples and profile these peptides within their network [2,3,4,5,6,7,8,9,10]. Accurate visualization and quantification of Ang peptides is of utmost importance to adequately study the RAS. Proteins from tissue sections have been analyzed, allowing for the localization of biological molecules to distinct regions of tissue [16,17], demonstrating the diversity and flexibility of MALDI-TOF analysis
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