Proteomic analysis of the synaptic plasma membrane fraction isolated from rat forebrain.

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.

[1]  D. Goldowitz,et al.  Preliminary analysis of the mouse cerebellum proteome. , 2002, Brain research. Molecular brain research.

[2]  P. Hanson,et al.  Neurotransmitter release — four years of SNARE complexes , 1997, Current Opinion in Neurobiology.

[3]  J R Yates,et al.  Mass spectrometry. From genomics to proteomics. , 2000, Trends in genetics : TIG.

[4]  Hanno Langen,et al.  The rat liver mitochondrial proteins , 2002, Electrophoresis.

[5]  M. Mann,et al.  Electrospray ionization for mass spectrometry of large biomolecules. , 1989, Science.

[6]  B. Kanner.  Active transport of gamma-aminobutyric acid by membrane vesicles isolated from rat brain. , 1978, Biochemistry.

[7]  A. Burlingame,et al.  Protein identification by in-gel digestion, high-performance liquid chromatography, and mass spectrometry: Peptide analysis by complementary ionization techniques , 2001, Journal of the American Society for Mass Spectrometry.

[8]  G. Friso,et al.  Analysis of proteins from membrane‐enriched cerebellar preparations by two‐dimensional gel electrophoresis and mass spectrometry , 1999, Electrophoresis.

[9]  M. Karas,et al.  Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. , 1988, Analytical chemistry.

[10]  E. Benzel,et al.  Essentials of Neurochemistry , 1994 .

[11]  Yoshiya Oda,et al.  Identification of activity‐regulated proteins in the postsynaptic density fraction , 2002, Genes to cells : devoted to molecular & cellular mechanisms.

[12]  M. Kennedy,et al.  Identification of Proteins in the Postsynaptic Density Fraction by Mass Spectrometry , 2000, The Journal of Neuroscience.

[13]  R. Burgoyne,et al.  Phosphorylation of Cysteine String Protein by Protein Kinase A , 2001, The Journal of Biological Chemistry.

[14]  Yingming Zhao,et al.  The Presynaptic Particle Web Ultrastructure, Composition, Dissolution, and Reconstitution , 2001, Neuron.

[15]  L. Prokai,et al.  Investigation of cytolysin variants by peptide mapping: enhanced protein characterization using complementary ionization and mass spectrometric techniques. , 2002, Rapid communications in mass spectrometry : RCM.

[16]  Pavel A. Pevzner,et al.  De Novo Peptide Sequencing via Tandem Mass Spectrometry , 1999, J. Comput. Biol..

[17]  V. Popov,et al.  Quantitative analysis of ultrastructural changes in synapses of the rat hippocampal field CA3 in vitro in different functional states , 1986, Neuroscience.

[18]  J. Yates,et al.  An automated multidimensional protein identification technology for shotgun proteomics. , 2001, Analytical chemistry.

[19]  F. Hajós.  An improved method for the preparation of synaptosomal fractions in high purity , 1975, Brain Research.

[20]  S. Grant,et al.  Proteomics in Neuroscience: From Protein to Network , 2001, The Journal of Neuroscience.

[21]  M. Karas,et al.  Suppression effects in enzymatic peptide ladder sequencing using ultraviolet ‐ matrix assisted laser desorption/ionization ‐ mass spectrometry , 1998, Electrophoresis.

[22]  John R Yates,et al.  Multidimensional separations for protein/peptide analysis in the post-genomic era. , 2002, BioTechniques.

[23]  L. Buck,et al.  Molecular Adaptations for Survival during Anoxia: Lessons from Lower Vertebrates , 2002, The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry.

[24]  H. Wenschuh,et al.  The dominance of arginine-containing peptides in MALDI-derived tryptic mass fingerprints of proteins. , 1999, Analytical chemistry.

[25]  J. DeGnore,et al.  Fragmentation of phosphopeptides in an ion trap mass spectrometer , 1998, Journal of the American Society for Mass Spectrometry.

[26]  G. Lubec,et al.  Rat brain proteins: Two‐dimensional protein database and variations in the expression level , 1999, Electrophoresis.

[27]  B. Soltys,et al.  Mitochondrial-matrix proteins at unexpected locations: are they exported? , 1999, Trends in biochemical sciences.

[28]  M. Chung,et al.  Proteome analysis of butyrate‐treated human colon cancer cells (HT‐29) , 2002, International journal of cancer.

[29]  D. Desiderio,et al.  Analysis of the human lumbar cerebrospinal fluid proteome , 2002, Electrophoresis.

[30]  S. Engelmann,et al.  Proteomics, DNA arrays and the analysis of still unknown regulons and unknown proteins of Bacillus subtilis and pathogenic gram-positive bacteria. , 2000, International journal of medical microbiology : IJMM.

[31]  S. Grant,et al.  Proteomic analysis of NMDA receptor–adhesion protein signaling complexes , 2000, Nature Neuroscience.

[32]  M. Cascio,et al.  Structural biology and structural genomics/proteomics. , 2002, The journal of peptide research : official journal of the American Peptide Society.

[33]  A. Morgan,et al.  Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I. , 2002, The Biochemical journal.