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An emerging way to study neuropsychiatric or neurodegenerative diseases is by performing proteomic analyses of brain tissues. Here, we describe methods used to isolate and identify the proteins associated with a sample of interest, such as the synapse, as well as to compare the levels of proteins in the sample under different conditions. These techniques, involving subcellular fractionation and modern quantitative proteomics using isotopic labels, can be used to understand the organization of neuronal compartments and the regulation of synaptic function under various conditions.
Neuroproteomics is the study of the proteome, or the collection of proteins encoded by the genes of an organism, in particular that of the central nervous system (CNS). With the development of new techniques and the improvement of those already available, it is now possible not only to identify proteins, but also to determine changes in their abundance under various conditions (1). This is particularly useful in understanding the physiological function of biological systems, as well as determining the functional implication of alterations in proteins in disturbed states, such as those induced by neurodegenerative disorders and/or drugs of abuse (2).
In the CNS, synapses are essential for the communication between neurons. Upon stimulation, a presynaptic neuron releases neurotransmitters that bind to receptors in the postsynaptic neuron, which in turn induces a series of events in response to the stimulus. One of the most fascinating properties of the CNS is synaptic plasticity, or the ability to reconfigure and/or modulate synapses to accommodate for the wide variety of stimuli that they receive at any given time. The inability to respond adequately may lead to the development of neurodegenerative or addictive disorders.
Neuroproteomic studies have started to identify the proteins present in different compartments of the synapse, including synaptosomes (3) and presynaptic and postsynaptic terminals (4), mainly through subcellular fractionation protocols. This type of approach facilitates the analysis by reducing the complexity of the system. Moreover, it enriches synaptic compartments with less abundant proteins, which are commonly masked by those with the highest abundance. One way to take advantage of this methodology is to select a brain region, isolate the synaptic fraction of interest before and after a treatment (such as exposure to morphine), and identify the proteins in the fraction as well as their relative changes upon treatment (5).
The approach described in this chapter is divided into two major sections. Section 2 describes the details for isolating a brain region and separating it into synaptic fractions, using subcellular fractionation. It is expected that (at least) two samples that undergo separate treatments (such as exposure to morphine and a control) are prepared. Section 3 details the steps required to isolate proteins from the fraction, digest them, differentially label them with appropriate isotopic labels, and perform mass spectrometry to identify the peptides and hence the proteins from the original sample. Details on data analysis are also covered, which results in the generation of a list of proteins and the relative change in protein levels upon treatment. While we have used specific examples from our research such as working with synaptic proteins after animal exposure to morphine, these protocols are general enough to work with other animal models, other brain regions, and a range of treatments. In addition, while specific fractionation and mass spectrometric equipment have been used, other instrumental platforms can be used with appropriate modifications.
This protocol is based on the use of rodent brain tissue. Once the animal is sacrificed by decapitation, the brain is rapidly removed and the regions of interest, such as striatum and hippocampus, are extracted (6).
Once the brain region of interest is collected, subcellular fractionation is performed to further simplify the sample for proteomic analysis. The following protocol allows the separation of various synaptic compartments, including synaptosomes, presynaptic and postsynaptic fractions (5, 7).
This protocol uses the Pierce® BCA Protein Assay for the protein estimation, which uses bicinchoninic acid for colorimetric detection and quantitation of proteins. It allows the detection of protein amounts ranging from 0.02 to 20 µg.
To determine protein amounts of the samples, it is necessary to use a common protein as a reference. This protocol uses bovine serum albumin (BSA) as the reference standard (see Table 1 for a guide in preparing the standards). The BSA stock used for the preparation of these curves has a concentration of 1mg/mL.
These instructions assume the use of a Mini-Protean II electrophoresis cell gel system. It is critical that the glass plates for the gels are scrubbed clean with a detergent after use and rinsed extensively with distilled water.
To validate the fractionation protocol, Western blotting analysis can be performed using the various aliquots obtained during the procedure. Antibodies for presynaptically- and postsynaptically-enriched proteins, such as Syntaxin 1 and PSD95 respectively, can be used to confirm the separation of the subcellular fractions (Figure 2). Since the quality of the sample is critical for accuracy of the data, it is recommended to perform this validation prior to proteomic analysis. The following protocol is based on the use of the Odyssey Infrared Imaging System; therefore, we suggest blocking the membrane in Odyssey Blocking Buffer.
The peptide separation and mass spectrometric analysis described here were performed using a capLC™ (Micromass, UK) system coupled to an HCTUltra--PTM Discovery system ion-trap mass spectrometer (Bruker) equipped with an electrospray ionization source. The Hystar program (Bruker) was used to define the HPLC and mass spectrometry methods that were used to develop the solvent gradient and MS tune method and integrate the instruments used (8). Other MS and LC systems can be used with appropriate modifications to these protocols (9–11).
This work is partially supported by the National Institute on Drug Abuse through Awards No. DA018310 and DA017940 to JVS.
1TFA is known to improve resolution of chromatographic separations through ion-pairing; however when used at >0.1%, TFA suppresses analyte ionization in a MS run. Formic acid is known to increase analyte ionization. Commercial solvents (Fischer Scientific, Pittsburg, PA) used in this study for sample clean-up and MS analysis have 0.1 % FA and 0.01 % TFA that is optimum for both ion pairing and ionization.
2Change the filter paper each time a different brain sample is dissected.
3Once an animal is sacrificed, brain dissection should be performed on ice as quickly as possible to avoid protein degradation.
4Clean dissection tools with 70% ethanol during dissection to avoid contamination of the samples.
5Everything must be kept on ice at all times, unless otherwise specified.
6We recommend using fresh solutions and buffers during the fractionation experiment. They may be prepared the day before the experiment starts and stored at 4°C.
7If larger quantities of tissue are used for the fractionation experiment, amounts of buffers and solutions should be increased in proportion to the amount of tissue.
8We recommend increasing the centrifugation time during the generation of the sucrose gradient to 4 h or more when using higher amounts of tissue. This allows for a better separation of the layers.
9When treating the synaptosomes with 1mM Tris-HCl buffer, pH 6, in order to pellet the synaptic junctions it may be necessary to split the volume of the synaptosomes into more than one centrifuge tube due to the limitation of the total volume that can be added to the tubes. This experiment assumes the use of the Sorvall RC 5C Plus centrifuge and the SS-34 rotor, which holds tubes with a maximum capacity of ~30 mL.
10Make sure to store the sample with acetone in an appropriate freezer. After covering the tube, make a small hole in the paraffin covering the tube to avoid accumulation of vapors from the acetone.
11Avoid freezing and thawing the samples too many times, as this can compromise the integrity of the proteins in the samples.
12It is important to prepare enough WR in order to add 200 µL to each reaction. The equation in this section allows you to determine the amount of solution needed for all the standards and unknowns. To account for pipetting errors, add one extra reaction to the calculations.
13The sample-to-WR ratio is approximately 1:8 (v/v), with a range of sample volume between 10 to 25 µ;L. Depending on the abundance of the proteins in the unknown samples, it might be necessary to make dilutions of the samples prior to adding the specific volume to the microplate.
14Increasing the incubation time and/or temperature can lower the detection level.
15Color development continues even after cooling to room temperature, although at a slower rate; therefore, reading of the plate should be performed as quickly as possible.
16The transfer protocol assumes the use of the Mini Trans-Blot cell transfer system.
17When using the Odyssey Infrared Imaging System, primary antibodies can be diluted in Odyssey Blocking Buffer with 0.1% Tween-20 and 0.01% NaN3. Optimum dilution depends on the antibody; therefore we suggest determining it individually for each antibody of interest.
18Two different fluorescently-labeled secondary antibodies can be used with the Odyssey System, the Cy5.5 and the IRDye800™. These secondary antibodies should be diluted in Odyssey Blocking Buffer with 0.1% Tween-20 and 0.01% NaN3, at a range of 1:2,000 to 1:10,000, although lower concentrations can be used to detect very small amounts of protein. The diluted secondary antibody can be stored at 4°C and reused.
19Assuming there are two treatment groups (e.g. morphine or saline treatment), the samples are paired so that one treatment group receives the heavy or light isotopic label (SA-D or SA-H) (12–14). Divide the samples so that each aliquot contains 10–30 µg of protein. Greater or lesser amounts of protein can be used with the appropriate scaling of reagents (15).
20When adjusting the pH of the sample solutions using 1M NaOH, use the same amount of solution from samples to be compared, i.e. maintain a constant volume between samples.
21Protein identification should be based on two or more tryptic peptides.
22Discard the ratio of peak pairs with overlapping peaks peptides originating from other “unknown peaks.”