Retention Time Mass-charge Ratio Pairs for Label-free Differential Analysis of Peptides

A simple LC-MS based methodology is presented that allows relative changes in the abundance of peptides in highly complex mixtures to be determined. The proposed method involves signal extraction, generation of features, alignment of corresponding features across different replicates/samples, and monitoring fold changes in relative abundance under different conditions without the use of isotopic or metabolic labeling strategies. By utilizing a nano-flow chromatographic separation system along with the high mass resolution and mass accuracy of an electrospray-ionization ion-trap mass spectrometer, one can make a quantitative comparison of thousands of ions emanated from identically prepared control and experimental samples. Using this configuration, we can determine the change in the relative abundance of a number of ions under the two conditions controlled solely by retention time and m/z value. By employing standard operating procedures for both sample preparation and LC-MS analysis, one typically obtains a retention time precision under 1.34 min, mass precision of 0.3 amu, and a median of quantitative variations less than 22%, as well as a good linearity of the response curve for peptide ions over 3 orders of magnitude in concentration. Highly selective identification of discriminatory peptides within a matrix of constitutive peptides using targeted MS/MS is achieved. The application of this method may represent a promising approach for quantitative peptidomics and discovery of peptide biomarkers.