Proceedings book download - 5Z.com

Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010A Neuropeptidomics Study of the Bovine Hypothalamus RevealsNovel Endogenous Peptides and Processing PathwaysMichelle L. Colgrave 1 *, Li Xi 1,2 , Sigrid Lehnert 1 ,Traute Flatscher-Bader 3,4 , Henrik Wadensten 5 , Anna Nilsson 5 ,Per E. Andren 5 , and Gene Wijffels 11 CSIRO Livestock Industries, 306 Carmody Rd, St Lucia, QLD 4067, Australia; 2 College ofVeterinary Medicine, Northwest A&F University, Xi'an, 712100, China; 3 CRC for BeefGenetic Technologies, C.J. Hawkins Homestead, University of New England, Armidale,New South Wales, 2351, Australia; 4 The University of Queensland, School of AnimalsStudies, Gatton, QLD 4343, Australia; 5 Department of Pharmaceutical Biosciences,Medical Mass Spectrometry, Biomedical Centre, P.O. Box 583, Uppsala University,SE-75123, Uppsala, SwedenIntroductionMass spectrometry (MS) has been adopted for neuropeptide research because of its abilityto rapidly and sensitively detect, characterize and quantify neuropeptides. Peptidomics isthe term coined to describe the field that deals with the comprehensive qualitative andquantitative analysis of peptides in biological samples [1]. While peptidomic studies holdconsiderable promise for the discovery of new bioactive molecules and the elucidation ofbiochemical regulatory networks, there are a number of challenges that remain to beresolved. The low concentration at which many endogenous peptides exist and are active iscompounded by the vast array of proteins present in biological samples at much higherconcentrations. Furthermore, neuropeptide processing in vivo begins with cleavage byprohormone convertases at dibasic sites and subsequent trimming of the basic residues bycarboxypeptidase D/E. The end result is peptides of small size that lack basic residues.These peptides may be difficult to ionize and/or display lower charge states in the massspectrometer. In the current study, we have evaluated a range of data acquisition strategiesin the investigation of snap-frozen bovine hypothalamus samples.Results and DiscussionIn order to maximize the number of peptide identifications, we employed six parallelacquisition strategies. These are detailed in Table 1 as Strategies A-F. Not surprisingly, thehighest number of identifications was achieved when twice the amount of sample wasloaded onto the HPLC column (Strategy F: 104 peptides).The use of the “Smart Exit” software feature has recently found utility in typicalproteomics experiments [2,3]. This feature works by monitoring the quality of the MS/MSspectra “on-the-fly” and, upon fulfilling the criteria for a “good” spectrum, exits itsacquisition thus shortening the information-dependent acquisition (IDA) cycle time andenabling more spectra to be acquired in the LC-MS timeframe. We used the SmartExitsoftware feature that enabled the acquisition of MS/MS spectra of varying quality – ineffect we achieved greater signal-to-noise (S/N) ratios for the fragment ions giving rise tohigher scoring peptide matches, but at the detriment of cycle time. The method utilizing theSmartExit software functionality in which the SmartExit setting was increased to themaximum value (20) to obtain the highest quality data for each MS/MS spectrum gave thenext highest number of peptide identifications (Strategy E: 74 peptides). In comparison, thenumber of identifications from the acquisition strategy using a ‘normal’ SmartExit setting(Strategy A: SmartExit 2) was lower (63 peptides). Surprisingly, the use of a mediumsetting (6) resulted in a decreased number of identifications (53 peptides). This can beexplained by the lower number of precursors selected for MS/MS (25% less). These dataindicated that the advantage of increasing spectral quality, through use of the SmartExitfunctionality, was only justified when using the highest setting.We also employed the dynamic background subtraction (DBS) software feature, whichenables the selection of relatively minor precursor ions in the presence of high backgroundconditions. In a typical LC-MS/MS analysis, there are a large number of potential candidateions at any given point in time. In a standard IDA experiment, the most intense ions areselected for MS/MS. Despite being abundant in nature, these ions might be from the456