retentiontimemasschargeratiopairsforlabelfreedifferentialanalysisofpeptides

A simple LC-MS based methodology is presented that allows relative changes in abundance of peptides In highly complex mixtures to he determined. Proposed method involves signal extraction, generation of features, alignment of corresponding features across different replicates/samples, and monitoring fold changes in relative abundance among different conditions without requiring the use of isotopic or metabolic labeling strategies. Utilizing a nano-flow chromatographic separation system along with the high mass resolution and mass accuracy of all electrospray-ionization (ESI) ion-trap mass spectrometer, the quantitative comparison of thousands of ions emanating front identically prepared control and experimental samples can be made. Using this configuration, the change in relative abundance of a small number of ions between the two conditions solely can I)e determined by retention time and m/z value. Employing standard operating procedures for both sample preparation and ESI-mass spectrometry, one typically obtains under 1.34 min relention time precision, 0.3 amu mass precision and a median of RSD 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 constitutively represented 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