RESEARCH ARTICLE Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures Guo-Zhong Li 1 , Johannes P. C. Vissers 2 , Jeffrey C. Silva 1 * , Dan Golick 1 , Marc V. Gorenstein 1 and Scott J. Geromanos 1 1 Waters Corporation, Milford, MA, USA 2 Waters Corporation, Manchester, UK A novel database search algorithm is presented for the qualitative identification of proteins over a wide dynamic range, both in simple and complex biological samples. The algorithm has been designed for the analysis of data originating from data independent acquisitions, whereby mul- tiple precursor ions are fragmented simultaneously. Measurements used by the algorithm include retention time, ion intensities, charge state, and accurate masses on both precursor and product ions from LC-MS data. The search algorithm uses an iterative process whereby each iteration incrementally increases the selectivity, specificity, and sensitivity ofthe overall strategy. Increased specificity is obtained by utilizing a subset database search approach, whereby for each subsequent stage of the search, only those peptides from securely identified proteins are queried. Tentative peptide and protein identifications are ranked and scored by their relative correlation to a number of models of known and empirically derived physicochemical attributes of proteins and peptides. In addition, the algorithm utilizes decoy database techniques for automatically determining the false positive identification rates. The search algorithm has been tested by comparing the search results from a four-protein mixture, the same four-protein mixture spiked into a complex biological background, and a variety of other “system” type protein digest mix- tures. The method was validated independently by data dependent methods, while concurrently relying on replication and selectivity. Comparisons were also performed with other commercially and publicly available peptide fragmentation search algorithms. The presented results demon- strate the ability to correctly identify peptides and proteins from data independent acquisition strategies with high sensitivity and specificity. They also illustrate a more comprehensive analysis of the samples studied; providing approximately 20% more protein identifications, compared to a more conventional data directed approach using the same identification criteria, with a con- current increase in both sequence coverage and the number of modified peptides. Received: July 3, 2008 Revised: October 16, 2008 Accepted: October 20, 2008 Keywords: Database searching / Data independent LC-MS / Multiplexed LC-MS / Parallel LC-MS / Shotgun sequencing 1696 Proteomics 2009, 9, 1696–1719 1 Introduction Traditional mass spectrometric approaches for the identifi- cation of peptides from enzymatically digested proteins Correspondence: Dr. Johannes P. C. Vissers, WatersCorporation, Atlas Park, Simonsway, Manchester M22 5PP, UK E-mail: hans_vissers@waters.com Fax: 144-161-435-4444 Abbreviations: DDA, data dependent analysis; MRM, multiple reaction monitoring; ROC, receiver operating characteristic * Current address: Cell Signaling Technology, Inc., 3 Trask Lane, Danvers, MA 01923, USA. DOI 10.1002/pmic.200800564  2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com