Uncovering Effects of Ex Vivo Protease Activity during Proteomics and Peptidomics Sample Extraction in Rat Brain Tissue by Oxygen-18 Labeling Christoph Stingl, † Marcus Sö derquist, ‡ Oskar Karlsson, § Mats Bore ́ n, ‡ and Theo M. Luider* ,† † Department of Neurology, Erasmus University Medical Center, 3000 CA Rotterdam, The Netherlands ‡ Denator AB, 413 46 Gothenburg, Sweden § Department of Pharmaceutical Biosciences, Uppsala University, 751 05 Uppsala, Sweden * S Supporting Information ABSTRACT: In biological samples, proteins and peptides are altered by proteolytic activity. The actual ex vivo form of the peptidome or proteome analyzed, therefore, does not always reflect the natural in vivo state. Sample stabilization and sample treatment are thereby decisive for how far these two states diverge. To assess ex vivo formation of peptides, we used enzymatic incorporation of oxygen-18 water during proteolysis (PALeO approach) to label ex-vivo-formed peptides in rodent brain tissue. Rates of ex-vivo-formed peptides were determined in 25 samples that were stabilized and treated by six different protocols, whereby samples were subjected to different conditions such as temperature, urea concentration, and duration of treatment. Samples were measured by nano LC-Orbitrap-MS, and incorporation of oxygen-18 was determined by MS/MS database search and analysis of the precursor isotope pattern. Extent of ex vivo degradations was affected relevantly by the sample treatment protocol applied and stopped almost completely by heat stabilization. Determination of the formation state by oxygen- 18 incorporation by MS/MS database search correlated well to more elaborate analysis of the MS isotope pattern. Overall, oxygen-18 labeling in combination with shotgun data-acquisition and MS/MS database search offers an adjuvant and easily applicable tool to monitor sample quality and fidelity in peptide and neuropeptide sample preparations. KEYWORDS: peptidomics, neuropeptides, oxygen-18, sample stabilization, quality control ■ INTRODUCTION When tissue is dissected and taken out of its natural environment, inherent biological processes, such as apoptosis or clotting, will continue in the ex vivo state of the tissue. These postsampling effects continue due to endogenous enzymatic activity resulting in changes to proteins and peptides. Later analysis of such a sample will show an overlap between in vivo state and ex vivo state confusing the analysis of proteins and peptides. Thus, an uncertainty is created whether some detected biomolecules are endogenously produced or formed by ex vivo degradation and results may be biased. These ex vivo alterations, if not inhibited at an early stage of the sample processing in an efficient and reproducibly way, further introduce significant variability in the proteomics analysis. 1 In the field of proteomics, various methods of protease activity inhibition are knownsuch as addition of specific protease inhibitors, heating or boiling, freezing, precipitation, or changes of pH, among othersand are used dependent to the sample type and proteomics application. 2,3 In the analysis of neuro- peptides in brain tissue, the prevention of general protein breakdown of cytosolic proteins is of utter importance. The utilization of effective stabilization methods such as microwave radiation or conductive heat stabilization enables identification of low-abundant neuropeptides and extends the total number of neuropeptides identified. 3-6 One way to determine the time-point of protease activity, in vivo or ex vivo, utilizes the incorporated of oxygen-18 ( 18 O) at the C-terminal carboxyl group of a peptide fragment. 7,8 The mechanism underlying this protease catalyzed reaction was described in detail by Schnö lzer et al. 8 In brief, during proteolysis, proteases bind to their substrate whereby the peptide bond is cleaved, and subsequently, the protease- substrate intermediate is hydrolyzed and water from the surrounding solvent system is incorporated. Further, some proteases, such as trypsin or Glu-C, recognized also the cleaved fragment as pseudosubstrate and will continue to bind and hydrolyze the C-terminal carboxyl-group of the fragment peptides, whereby further water from the solvent system is incorporated. If the surrounding water contains oxygen-18, products from both reactions will result in incorporation of oxygen-18, whereby the extent of C-terminal oxygen-18 incorporation is in equilibrium with the ratio of H 2 18 O in the surrounding water. The number of incorporated oxygen-18 Received: December 12, 2013 Article pubs.acs.org/jpr © XXXX American Chemical Society A dx.doi.org/10.1021/pr401232e | J. Proteome Res. XXXX, XXX, XXX-XXX