An Integrated, Directed Mass Spectrometric Approach for In-depth Characterization of

LC-MS/MS has emerged as the method of choice for the identification and quantification of protein sample mix-tures. For very complex samples such as complete pro-teomes, the most commonly used LC-MS/MS method, data-dependent acquisition (DDA) precursor selection, is of limited utility. The limited scan speed of current mass spectrometers along with the highly redundant selection of the most intense precursor ions generates a bias in the pool of identified proteins toward those of higher abun-dance. A directed LC-MS/MS approach that alleviates the limitations of DDA precursor ion selection by decoupling peak detection and sequencing of selected precursor ions is presented. In the first stage of the strategy, all detect-able peptide ion signals are extracted from high resolution LC-MS feature maps or aligned sets of feature maps. Th