Considering the observed electron density at the synapse and the fact that active zones deter-mine not only the site and but also the timing for synaptic transmission, the number of known active zone proteins is surprisingly low. In comparison to the PSD, it is believed that the com-position of presynaptic nerve terminal is only party uncovered yet. In order to fully understand the mechanisms that regulate the formation, maintenance and function of neurotransmitter re-lease, it is necessary to reveal the exact protein composition of the active zone and analyze the interactions.
Due to the fact that proteomic studies mainly failed, because presynaptic preparations were scarce and insufficient, the main goal of this thesis was to develop an isolation protocol for a presynaptic fraction that allows for comprehensive proteomic studies. This protocol required an efficient removal of the postsynaptic density from synaptosomes, a challenging task that engaged a significant part of this work. Based on this protocol I wanted to validate presynaptic candidates and identify novel molecular players that are required for the docking of synaptic vesicles to the plasma membrane. By performing state-of-the-art quantitative proteomics, I hope to discriminate true presynaptic proteins from other contaminations. With this method I additionally wanted to describe changes in the presynaptic proteome in response to biological perturbations since this has not been done for the presynaptic site. Such an applicational ex-ample will hopefully provide a basis for further similar studies that will help to understand the mechanisms of synaptic transmission.
2.1 Materials
2.1.1 Chemicals
Standard chemicals used in this study were obtained from either Sigma-Aldrich (Steinheim, Germany), Roth (Karlsruhe, Germany), Merck (Darmstadt, Germany), Boehringer (Ingelheim, Germany), Fluka (Buchs, Germany), Serva (Heidelberg, Germany), Roche (Basel, Switzer-land) or Waters (Eschborn, Germany). All chemicals were of at least analytical purity. Other chemicals are listed below (2.1).
Chemical Source
Pefabloc Roche
Pepstatin Peptide institute
Phenylmethylsulfonylfluorid (PMSF) Roth
Eupergit C1Z beads Roehm Pharma
GTPγS Roche
GDP Sigma Aldrich
Ni-NTA Agarose Qiagen
5 ml MonoQ column Amersham
RapiGest Waters
Triethylammonium bicarbonate (TEAB) Sigma Aldrich
Trifluoracetic acid Sigma Aldrich
Formic acid Fluka
Triton-X-100 Sigma-Aldrich
Triton-X-114 Sigma-Adrich
Dulbecco’s Modified Eagle’s Medium (DMEM) Lonza
Penicillin/Streptomycin Lonza
Table 2.1: Chemicals used in this study.
15
2.1.2 Enzymes
The enzymes that were used in this study are listed in 2.2 and were obtained from Fermen-tas (St. Leon-Rot, Germany), New England Biolabs (NEB; Ipswich, MA, USA), Promega (Madison, WI, USA) or Roche (Basel, Switzerland). All restriction enzymes, ligases and poly-merases were used according to manufacturer’s instructions (including the supplied buffers).
Enzyme Application Source
Proteinase K Synaptosome digest Roche
Trypsin Synaptosome digest Roche
Trypsin (sequence grade modified) In-solution digest for MS Promega
Restriction enzymes DNA digest NEB or Fermentas
Ligase DNA ligation NEB
Pfu polymerase Polymerase chain reaction Promega
Table 2.2: Enzymes used in this study.
2.1.3 Kits
The commercially purchased kits used in this study are listed in 2.3 and were used for the stated application according to manufacturer’s instructions (including the supplied buffers).
Kit Application Source
Western LighteningTMPlus-ECL Chemoluminescence detection Perkin Elmer PierceRBCA Protein assay protein quantification ThermoFisher LipofectamineTM2000 transient cell transfection Invitrogen NucleoBondRXtra Plasmid purification (preparative scale) Macherey-Nagel NucleoSpinRPlasmid Plasmid purification (analytical scale) Macherey-Nagel
NucleoSpinRExtract DNA clean-up Macherey-Nagel
iTRAQTMreagent multiplex Kit quantitative peptide labeling Applied Biosystems
Table 2.3: Kits used in this study.
2.1.4 Antibodies
Antibodies used in this study are listed in Table 2.4. Antibodies were either generated in this laboratory or purchased at Abcam (Cambridge, UK), BD Bioscience (Erembodegem, Belgium), BioRad (Hercules,CA, USA), Jackson Immunoresearch Europe (Newmarket, UK), NeuroMab (Davis, USA), Synaptic Systems (Göttingen, Germany).
aterials17
Antibody Species Epitope Application Source
Synaptophysin 7.2 mouse monoclonal, affinity purified cytoplasmic tail WB (1:1000), IP [154]
Synaptophysin G96 rabbit polyclonal, serum cytoplasmic tail IF (1:200) [154]
Synaptobrevin 69.1 mouse monoclonal, ascites SATAATVPPAAPAGEG WB (1:2000) [155]
Munc18 rabbit polyclonal, serum full length WB (1:1000)
Munc13 mouse, monoclonal, affinity purified aa 3-317 WB (1:1000) Synaptic Systems
Piccolo rabbit polyclonal, affinity purified aa 439 - 4776 WB (1:500), IF (1:100) Synaptic Systems
Bassoon rabbit polyclonal, serum C-terminus WB (1:500) Synaptic Systems
Synaptotagmin 41.1 mouse monoclonal, ascites cytoplasmic domain WB (1:1000), IF (1:100) [156]
PSD95 mouse monoclonal, affinity purified aa 77-299 WB (1:2000), IF (1:200) NeuroMab
Homer rabbit polyclonal, affinity purified aa 1-186 WB (1:1000) Synaptic Systems
Syntaxin1A 78.2 mouse monoclonal, ascites N-terminus WB (1:1000), IF (1:100) [46]
NMDA receptor mouse monoclonal, ascites aa 660-811 WB (1:1000) [157]
AMPA receptor rabbit polyclonal, affinity purified C-terminus aa 826-906 WB (1:1000) Synaptic Systems
Na+/K+ATPase alpha 1 mouse monoclonal, ascites not known WB (1:2000) Abcam
SDHA mouse monoclonal, affinity purified not known WB (1:2000), IF (1:200) Abcam
Neuroligin rabbit polyclonal, affinity purified extracellular aa 46-165 WB (1:1000) Synaptic Systems
RIM mouse monoclonal, affinity purified aa 602-723 WB (1:500) BD Biosciences
VGlut1 rabbit polyclonal, serum C-terminus aa 456-560 WB (1:1000) [158]
Mint1 rabbit polyclonal, affinity purified aa 2-265 WB (1:1000) Synaptic Systems
CASK mouse monoclonal, affinity purified aa 318-415 WB (1:1000) NeuroMab
ERC 1b/2 rabbit polyclonal, affinity purified CDQDEEEGIWA WB (1:1000) Synaptic Systems
GFP rabbit polyclonal, serum full length WB (1:10000) Synaptic Systems
SynCAM rabbit polyclonal, affinity purified aa 167-181 WB (1:1000) Synaptic Systems
mouse IgG (Cy2 or Cy3 labeled) goat polyclonal, affinity purified IgG (H+L) IF (1:400) Jackson Immunoreserach rabbit IgG (Cy2 or Cy3 labeled) goat polyclonal, affinity purified IgG (H+L) IF (1:400) Jackson Immunoresearch
mouse IgG (HRP labeled ) goat polyclonal IgG (H+L) WB (1:2000) BioRad
rabbit IgG (HRP labeled) goat polyclonal IgG (H+L) WB (1:2000) BioRad
Table 2.4: Antibodies used in this study: IF (Immunfluorescence), WB (Western Blot), IP (Immunoprecipitation). Dilutions are marked in brackets.
2.1.5 Buffers and media
Buffer/media Composition
PBS 2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, 8 mM
Na2HPO4, pH7.3
TBST 15 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5 % (v/v) Tween 20 SDS running buffer 25 mM Tris-HCl, 192 mM Glycine, 0.1 % SDS
Transfer buffer 200 mM Glycine, 25 mM Tris, 0.04 % SDS, 20 % Methanol Homogenization buffer 320 mM sucrose, 5 mM Hepes, pH 7.4
Sodium buffer 10 mM Glucose, 5 mM KCl, 140 mM NaCl, 5mMNaHCO3, 1 mMMgCl2, 1.2 mMNa2HPO4, 20 mM HEPES pH 7.4
1x IP buffer 1x PBS, 5 mM Hepes pH 8.0, 3 mg/ml BSA 2x IP buffer 2x PBS, 5 mM Hepes pH 8.0, 6 mg/ml BSA
Cell culture media DMEM, 10 % FCS, 4 mM glutamine, 100 U/ml penicillin and streptomycin
Luria-Bertani medium (LB) 10 g tryptone, 5 g yeast extract and 10 g NaCl per 1L
Table 2.5: Buffers and composition that were regularly used in this study.
2.1.6 Mammalian cell lines and bacterial strains
The Human Embryonic Kidney 293 cell line (HEK293) were used for over-expression stud-ies. E.coli DH5α strains were used for molecular cloning and E.coli BL21 (DE3) for protein expression.
2.1.7 DNA constructs
The plasmid encoding JB1 was synthesized and purchased from GENEART (Regensburg, Ger-many) according to the sequence obtained from NM_001108129. Codon usage was optimized for mammalian expression systems. The plasmid encoding GDP-dissociation inhibitor GDI (R.
norvegicus) was a kind gift from Dr. Nathan Pavlos (University of Western Australia, Perth, Australia).