The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.
The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to naïve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment.
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/naïve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.
学习是基于记忆痕迹及其维护的形成。它已被广泛接受,一个潜在的机制可以表示神经元之间的现有突触联系的新的和/或重排的活性依赖性形成。在分子水平上,各种蛋白质修饰,亚细胞relocalizations和突触蛋白的周转变化已经描述1-4(Lamprecht的,2004#8)。然而,大多数的研究迄今针对选定的蛋白质,而不是对全球但复杂的突触蛋白质组合物。本方法允许一个学习实验后小鼠脑区突触蛋白质组变化公正的审查。合适的是使突触架构的学习诱导重组时间点相关的分子的快照。所描述的工作流程,需要不同的专家在动物行为,蛋白质生物化学,质谱和bioi一个特定的团队合作nformatics。
所选择的学习范例, 即频率调制的音调鉴别(FMTD),是在啮齿类动物5充分表征听觉辨别任务。学习和长期记忆的形成这个梭箱GO / NO-GO-任务涉及取决于增加皮质多巴胺信号和蛋白质合成机制。因此,最近对沙土鼠和小鼠的蛋白质组学研究揭示多巴胺和皮质突触部件的学习诱导塑料重排,而且在更基础的大脑区域FMTD学习和记忆6-8期间,理应相互作用。这表明,记忆的形成涉及不同脑区之间复杂的相互作用,因此,可能在蛋白质水平这些区域内差异调节。因此,选择的皮层和皮层下小鼠的大脑区域的解剖被包括在工作流程。
此外,可靠characterizati在偶数的突触蛋白组合物弱变化需要前和突触后隔室的富集而非匀浆或粗膜部分9的分析。蛋白质组学分析之前因此,突触体的制备利用建立的协议,以增加检测电平与动态范围为特定突触蛋白10,11进行说明。
使用无标签的高分辨率质谱定量目的的基本前提是高度的蛋白质样品的相似性。由于突触蛋白质组成,而细微的变化,预计学习,无标签的方法将是适当的比较,从相应的培训和天真的小鼠获得的蛋白样品后出现。可替代地,使用稳定同位素( 例如 TMT,iTRAQ的,ICPL和SILAC)以及基于MS2-标签自由QUA蛋白/肽的特定条件下的标签的策略ntification(SWATH)是有用的,但它们比选择无标记方法更昂贵或需要特别的质谱硬件。
因为蛋白质组掩护往往产生复杂的数据集,建议适当的数据解释生物信息的处理。其他荟萃分析可以支持更好地了解潜在的范式相关的变更,涉及的关键细胞过程和信号通路的识别潜在的分子机制。适当的方法也被描述如下。
这项研究提出了学习和小鼠不同脑区记忆的巩固过程中的突触蛋白表达变化的准确的定量分析进行优化的方法流程。该装置提供了学习的机会,在一个动物的水平蛋白的表达,尽管每个样品至少有三个技术重复进行质谱分析所需的应用程序的。
该方法考虑到前和postsynapse选自高分子量支架蛋白的特定的蛋白质组合物也是重要的介体蛋白承载介质或更低的分子量。突触制剂的在溶液消化导致有效地产生,因此,支架衍生肽的过表达。这,反过来,可能抑制较小或低丰度蛋白的分析。从SDS-PAGE馏分的建议制备每个样品与平行的在凝胶内消化过程合并的等分试样促进中,低丰度蛋白质的分析和表示高度推荐的互补方法。后从样品衍生的所有级分的分离质谱应用( 例如 ,在溶液消化,凝胶内消化,结合磷富集馏分)可以结合在相应的MS / MS数据集,并进一步用于蛋白质鉴定和量化由PEAKS计算软件或替代流行的软件包。
可替代地,在溶液中消化的样品的产生的样品的凝胶内消化衍生馏分(样品泳道分开处理的凝胶区域)和级分的个人应用(通过离子交换色谱,例如 ),以质谱可以增加分析深度。然而,这种延长的工作流显着地增加对于LS-MS / MS数据采集所需的时间。对于generatio学习和记忆形成的蛋白质组分析的指定时间期间的突触蛋白的重排的详细分子序列的n个是必需的。这个时候当然会,直到动物的性能达到学习曲线的渐近水平约后后甚至第一堂训练课中立即启动,涵盖了近网状的时间框架。 8 – 10天的培训( 见图2细节)。
突触蛋白的磷酸化的变化分析需要FMTD学习过程中特别注重所选择的时间框架。在启动已知由蛋白质磷酸化dephosphorylations触发突触蛋白重组,一方面信号通路有望在动物训练的早期阶段。另一方面,也有其调节S内的连接和装配已知多种磷酸突触蛋白的长期持久的修改ynaptic结构19,20。这些翻译后修饰甚至在记忆巩固稍后的时间点的预期。
该蛋白质组的工作流程产生的复杂的数据集需要处理生物信息学,以确定参与的分子途径和关键分子。荟萃分析显示显著任职人数过多的途径,从而起到在学习和记忆过程中发挥作用。
The authors have nothing to disclose.
We wish to thank Yvonne Ducho and Kathrin Pohlmann for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 779) and by the State Saxony-Anhalt / European Regional Development Fund (ERDF) via the Center for Behavioral Brain Sciences (CBBS).
3M Empore Solid Phase Extraction- Filter | 3M Bioanalytical Technologies | 4245SD | 7 mm/3 ml |
Acclaim PepMap 100 | Dionex/Thermo Scientific | 164564 | 100 µm x 2 cm, C18 |
Acclaim PepMap 100 | Dionex/Thermo Scientific | 164569 | 75 µm x 25 cm, C18 |
Acetic acid | Carl Roth GmbH | 3738.1 | |
Acetonitrile (ACN) | Carl Roth GmbH | AE70.2 | |
Acrylamide (30%) | AppliChem | A0951 | |
Ammonium hydrogen carbonate | Fluka | 9830 | |
Ammonium hydroxide | Fluka | 44273 | |
Ammonium persulfate (APS) | AppliChem | A2941 | |
Biofuge pico | Heraeus GmbH | 75003280 | |
Blue R-250 | SERVA Electrophoresis GmbH | 17525 | |
Bromophenol Blue | Pharmacia Biotech | 17132901 | |
C57BL/6J mice | Charles River | ||
Cantharidin | Carl Roth GmbH | 3322.1 | |
Centrifuge tubes for MLS-50 | Beckman Coulter | 344057 | |
Centrifuge tubes for TLA 100.1 rotor | Beckman Coulter | 343776 | |
Dithiothreitol (DTT) | AppliChem | A1101 | |
Eppendorf 5417R centrifuge | VWR | 22636138 | |
Eppendorf A-8-11 rotor | VWR | 5407000317 | |
Formic acid | Fluka | 14265 | |
GeneCodis | http://genecodis.cnb.csic.es/ | ||
Gephi | https://gephi.org/ | ||
Glycerol | AppliChem | A1123 | |
Glycine | AppliChem | A1067 | |
HALT Phosphatase Inhibitor Cocktail | Pierce /Thermo Scientific | 78420 | |
HEPES Buffer solution | PAA Laboratories GmbH | S11-001 | |
Homogenization vessel 2 mL | Sartorius AG | 854 2252 | |
Hydrochloric acid | Sigma-Aldrich | H1758 | |
Imidazole | Sigma-Aldrich | I2399 | |
Ingenuity Pathway Analysis | Qiagen | ||
Iodoacetamide (IAA) | Sigma-Aldrich | I1149 | |
Laboratory drilling drive K-ControlTLC 4957 | Kaltenbach & Vogt GmbH | 182997 | |
LTQ Tune Plus 2.7.0.1112 SP2 | Thermo Scientific | ||
LTQ Orbitrap Velos Pro | Thermo Scientific | ||
Macs-mix tube rotator | Miltenyi Biotech | 130-090-753 | |
Magic Scan 4.71 | UMAX | ||
Methanol | Carl Roth GmbH | AE71.2 | |
MLS-50 rotor | Beckman Coulter | 367280 | |
Optima MAX Ultracentrifuge | Beckman Coulter | 364300 | |
PageRuler Prestained Protein Ladder | Thermo Scientific | 26616 | |
PEAKS 7.5 | Bioinformatic Solutions | ||
Phosphatase Inhibitor Cocktail 3 | Sigma-Aldrich | P0044 | |
PhosphoRS 3.1 | IMP/IMBA/GMI | ||
PhosSTOP | Roche | 4906845001 | |
Plunger/pestle made of PTFE | Sartorius AG | 854 2651 | |
PotterS homogenizer | Sartorius AG | 853 3024 | |
Protease Inhibitor complete mini | Roche | 4693159001 | |
Quantity One 4.5.1 | BioRad | ||
RapiGest | Waters | 186002122 | |
Shuttle box | Coulbourne Instruments | ||
Sodium dodecylsulfate (SDS) | AppliChem | A1112 | |
Sodium molybdate | Carl Roth GmbH | 274.2 | |
Sodium tartrate dihydrate | Sigma-Aldrich | 228729 | |
SONOREX RK 156 Ultrasonic Bath | BANDELIN electronic GmbH & Co. KG | 305 | |
Soundproof chamber | Industrial Acoustics Company | ||
Sucrose | Carl Roth GmbH | 4621.2 | |
Tetramethyl ethylene -1,2-diamine (TEMED) | Sigma-Aldrich | T9281 | |
Thermomixer basic | CallMedia | 111000 | |
Titansphere TiO 5µm | GL Sciences Inc. Japan | 502075000 | |
TLA 100.1 rotor | Beckman Coulter | 343840 | |
Trifluoro acetic acid (TFA) | Sigma-Aldrich | T6508 | |
Tris ( hydroxymethyl) aminomethane (TRIS) | AppliChem | A1086 | |
Triton X-100 | Sigma-Aldrich | T8532 | |
Trypsin Gold | Promega | V5280 | |
Ultimate 3000 Ultra HPLC | Dionex/Thermo Scientific | ||
Ultracentrifuge tube | Beckman Coulter | 343776 | |
Unijet II Refrigerated Aspirator | Uniequip Laborgeräte- und Vertriebs GmbH | ||
UNIVAPO 100 H Concentrator Centrifuge | Uniequip Laborgeräte- und Vertriebs GmbH | ||
Urea | AppliChem | A1049 | |
Water (high quality purifed) | Resistivity: > 18.2 MΩ*cm at 25 °C Pyrogens: < 0.02 EU/ml TOC: < 10 ppb | ||
Xcalibur 3.0.63 | Thermo Scientific | ||
ZipTipC18 Pipette Tips | MILLIPORE | ZTC18S960 |