Molecular Brain Research 117 (2003) 116–128 www.elsevier.com / locate / molbrainres Research report Proteomic analysis of the synaptic plasma membrane fraction isolated from rat forebrain a a a,b, * Stanley M. Stevens Jr. , Alevtina D. Zharikova , Laszlo Prokai a Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610-0485, USA b The McKnight Brain Institute, University of Florida, Gainesville, FL, USA Accepted 17 June 2003 Abstract Mass spectrometry (MS) in conjunction with liquid chromatography and gel separation techniques has been utilized to identify synaptic plasma membrane (SPM) proteins isolated from rat forebrain and digested with the protease trypsin. Initial results employing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation of the SPM protein mixture have shown that several membrane proteins were under-represented due to solubilization problems in the dimension of isoelectric-point focusing. Given the complexity of the SPM, multiple stages of separation were necessary prior to mass spectrometric detection in order to facilitate protein identification. This particular study involved several approaches using one-dimensional (1D) sodium dodecyl sulfate (SDS)-PAGE, strong cation-exchange (SCX) chromatography and capillary reversed-phase high performance liquid chromatography (HPLC) techniques. In addition to these gel and HPLC separation stages, complementary information was obtained by using both matrix-assisted laser desorption / ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Data-dependent acquisition employing capillary HPLC-nanoESI / MS allowed for the detection of low-abundance tryptic peptides in the digested SPM fraction and identification of the corresponding proteins when product-ion information of a single or multiple peptides was used in protein database searching. The potential value of this subproteome methodology was exemplified by the identification of several proteins relevant to synaptic physiology which included various transporters, receptors, ion channels, and enzymes.  2003 Elsevier B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Gene structure and function: general Keywords: Rat forebrain; Proteomics; Mass spectrometry; Synaptic membrane 1. Introduction logical challenge [13]. At present, analysis of protein constituents in organelles and specifically isolated (e.g. A large number of proteins and their complex networks immunoblotted) subcellular complexes appears to be a covering diverse biological functions can be studied via the viable (‘subproteome’) approach that reduces complexity emerging field of proteomics [24]. While mapping of all and allows for a meaningful application of the technique to proteins and their intricate interplay in prototype uni- neuroscience [17]. cellular eukaryotes are being pursued extensively by Surprisingly, proteomics studies on the organization of various techniques [35], the anatomical and cellular com- protein networks in the synapses, which represent the plexity of the mammalian central nervous system with its principal means of communication among neurons [33], vast number of synapses represents a technical and bio- have been scarce [13,32]. A probable reason is that integral parts of the synaptic protein networks are membrane or membrane-bound proteins such as receptors, transport *Corresponding author. Present address: Department of Medicinal channels, ectoenzymes, etc., prone to defy efforts for their Chemistry, College of Pharmacy, University of Florida, Gainesville, FL identification by two-dimensional (2D) gel electrophoresis 32610-0485, USA. Tel.: 11-352-392-3421; fax: 11-352-392-3421. followed by in-gel protease (trypsin) digestion, matrix- E-mail addresses: lprokai@grove.ufl.edu (L. Prokai), http: / / www.cop.ufl.edu / prokai / (L. Prokai). assisted laser desorption / ionization (MALDI) [19] / time- 0169-328X / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0169-328X(03)00282-1