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Table 1. Identification of neuropeptides using six acquisition strategies. The conditions usedfor data analysis are shown in the top half of the table, while the results obtained are detailedin the bottom half of the table. A total of 118 neuropeptides were identified in the stabilizedsamples. DBS refers to the Dynamic Background Subtraction software feature and SmartExitrefers to the quality of the MS/MS spectra obtained where the value may be 1-20.Strategy A B C D E FInjection volume (µl) 5 5 5 5 5 10Precursor z 2-5 2-5 2-5 1-3 2-5 2-5DBS Off Off On On Off OffSmartExit 2 6 2 2 20 6No. neuropeptide ID’s 63 53 53 54 74 104% of total (118) 53 45 45 45 62 87No. spectra acquired 973 742 423 877 900 1210% spectra identified 57 55 68 36 68 76background, i.e., present for the duration of the LC-MS analysis, or they may be from apeptide derived from a highly abundant protein. Many of the less intense ions may avoidselection for MS/MS acquisition. The DBS software feature measures the intensity of eachion over several scans and triggers MS/MS acquisition based at the point in time at whichthey are rising most quickly in intensity. The use of the Dynamic Background Subtraction(DBS) feature resulted in a decreased number of peptide identifications (Strategy B: 53 forprecursors with charge state 2-5; 54 for precursors with charge state 1-3). The DBStriggereddatasets of multiply charged precursors showed a much lower number ofprecursors selected for MS/MS (approximately half of the number selected in non-DBSruns). For the DBS-triggered datasets in which singly charged precursors were included(Strategy D), the number of MS/MS spectra acquired was roughly equivalent to thatobtained by Strategies A and E, but the percentage of identified spectra dramaticallydecreased.The acquisition strategy used, specifically focusing on acquisition of MS/MS spectraof singly to triply charged precursors rather than precursors of charge +2 to +5 (as is usedin our traditional proteomic workflows) did not lead to a significant increase in the numberof neuropeptide identifications despite the increased number of spectra acquired. Manualinspection of the spectra provided by Strategy D proved that the vast majority of singlycharged precursors selected were not peptidic in nature and/or yielded poor MS/MSspectra.In conclusion, the optimization of the acquisition conditions and the databasesearching strategy resulted in increases in the number of neuropeptides identified (up to2-fold increase) with >75% of spectra identified.AcknowledgmentsWe thank Alun Jones and the Molecular and Cellular Proteomics Facility at the University ofQueensland Institute for Molecular Bioscience for access to mass spectrometry instrumentation. Wethank the Cooperative Research Centre for Beef Genetic Technologies for making tissue samplesavailable to this project, and the Kilcoy Pastoral Company for their cooperation in sample collections.Professor Michael D’Occhio developed the bovine hypothalamus sampling protocol and Dr. AinuSuhaimi was involved in bovine hypothalamus sample collection.References1. Schulz-Knappe, P., et al. Comb. Chem. High Throughput Screen. 4, 207-217 (2001).2. Zhu, M., et al. J. of Proteomics 73, 790-805 (2010).3. Dwivedi, R.C., et al. J. Proteome Res. 9, 1144-1149 (2010).457