There are several steps for successful 2-DE analysis. Sample preparation Speaking of sample preparation, the native samples need to be converted to a physicochemical state suitable for the first dimension IEF and keep the native charge and Mr of the constituent proteins.
In order to get good results, appropriate sample preparation is essential. The sample preparation is various because of the difference of the types and origins of proteins. Ideally, the process will result in the complete solubilization, disaggregation, denaturation, and reduction of the proteins in the sample.
Proteins are amphoteric molecules and the positive, negative, or zero net charge they carry depending on the pH of the surroundings.
The isoelectric point pI is defined as the pH of a solution at which the net charge of the protein becomes zero. A protein with a positive net charge will migrate toward the cathode, becoming less positively charged until reaching its pI.
While a protein with a negative net charge will migrate toward the anode, becoming less negatively charged until it also reaches its pI.
To be specific, a protein mixture is loaded at the basic end of the pH gradient gel. After applying an electric field, the proteins are separated depending on charges, focusing at positions where the pl value is equivalent to the surrounding pH. Larger proteins will move more slowly through the gel, but with sufficient time will catch up with small proteins of equal charge. This method often contains four steps, including preparation of the gel, the equilibrium of the immobilized pH gradient IPG strips in SDS buffer, placing the equilibrated IPG strip on the SDS gel, and finally handling the electrophoresis.
SDS can make proteins denaturing and bind to the backbone at a constant molar ratio. When applying SDS and a reducing agent, like a DTT which can cleave disulfide bonds, proteins unfold into linear chains with negative charge proportional to the polypeptide chain length. As a result, this step requires a great deal of sample-specific optimization to avoid bias and contamination.
At last, your sample is solubilized! Where IEF gets more exciting is that the gel incorporates a pH gradient, and each protein moves only until it reaches its isoelectric point pI. The pI is the pH where a protein has no net charge, meaning the field has no effect and the protein stays put, focusing tightly into a band within 0. As straightforward as this sounds, IEF throws several wrenches into the scientific works.
First, proteins become less soluble and can even precipitate out as they move closer to their pI, especially in low-salt, IEF-friendly buffers. Second, IEF gels and buffers interfere with sample prep for mass spectrometry MS and can be difficult to stain for analysis. Finally, the second dimension! But first, those beautifully-focused proteins need to be solubilized again in SDS before they can be separated by their molecular weights on an orthogonal second axis.
Other than this equilibration and some additional care with timing and voltages, this step uses molecular weights to separate proteins through the tried-and-true SDS-PAGE method. At the end, the gel will have the proteins aligned along two axes: isoelectric point vs. This final step depends on your particular experimental endpoints: are you comparing protein expression? When comparing protein expression across different experimental samples, the gels are typically stained with silver or Coomassie blue for total protein.
Various image analysis platforms are then used to scan and compare the location and intensities of the separated proteins. Naturally, analysis is not as clean and simple as slapping a gel into the scanner. To improve reproducibility between samples, gels should be run in parallel, which can lead to logistic and diplomatic challenges in a lab with only one power supply and an imminent lab meeting.
Furthermore, interpreting that Morse-like pattern of dots and splotches requires protein identification and annotation beforehand, either through in-house efforts or via sweet, sweet annotated databases of similar samples. There are two general options for those in the protein identification phase. Gels were stained with the MS-compatible silver staining procedure [ 14 , 21 ]. The methods used for staining are summarized in Table 2.
The main aim of proteomics consists in the identification of differentially expressed proteins. To proceed with the 2DE analysis, the first step is the digitalization of gel images. Subsequent analysis was performed using the Image Master 2D-Platinum software, version 6. Briefly, raw images were imported into the software and processed for protein detection. The spot auto-detect function was used for all group comparisons applying the following parameters: smooth 2, min area 5, saliency 1.
Digital images were checked to ensure that all the proteins present in the gel were correctly detected. Using this option, the data becomes independent variables caused by differences in loading or staining [ 22 ]. Analysis was done using three independent replicates. Protein levels in every data set were compared to control group using unpaired t-test. Excel spreadsheet Microsoft was used for data plotting [ 23 ]. A main issue during spot excision and digestion procedure is avoiding contamination with keratins that can affect subsequent mass spectrometry analysis.
Proper lab-equipment and dedicated reagents mast be used for protein spot digestion. Protein spots, with a differential expression profile, were manually excised from silver stained gel using OneTouch Plus Spotpicker Gel company. Gel pieces were washed with Milli-Q water and destained incubating with a destaining solution Table 1 for 15 min.
Trypsin solution was carefully recovered and placed in a fresh tube t. The solution was recuperated and pooled in the fresh tube t. Peptide solution was concentrated using a speedy vac [ 24 , 25 ]. The analysis was performed as service in our mass spectrometry facility.
Purified peptides were re-suspended with 0. A binary gradient was used for the elution of peptides. Mass analysis was done by means of a quadrupole-orbitrap mass spectrometer Q-Exactive Thermo Fisher Scientific, Bremen, Germany working in positive ion mode, using nanoelectrospray nESI potential at V.
Precursor ion isolation was done using a mass window of 2. Dynamic exclusion was set at 15 s. Data were processed using Proteome Discoverer 1. We consider only protein hits generated with two successful peptide identifications valid and acceptable [ 27 ]. Western blot analysis of prohibitin and gelsolin was done to verify 2D gel electrophoresis data. The signal was detected using the specific horseradish peroxidase-conjugate secondary antibodies; blots were developed using the SuperSignal West Femto ECL substrate Pierce.
Densitometric software Alliance 2. Unpaired t-test was used to compare protein levels in each data set. BRCA1 is a tumor suppressor gene, often mutated in hereditary breast and ovarian cancers.
The gene encodes for a large protein involved in several cellular pathways that include DNA damage-induced cell cycle checkpoint activation, maintenance of genomic stability, DNA damage repair, as well as chromatin remodeling, protein ubiquitination, transcriptional regulation, and apoptosis [ 29 ]. Recently, it has been also reported that BRCA1 is able to induce reprogramming of metabolism toward aerobic glycolysis in breast cancer cells [ 30 ].
Transient knock down of BRCA1 was done at 48 h according to our previous results [ 20 ]. The analysis workflow is summarized in Figure 2. The analysis was performed using three biological replicates. Gene interference. Western blot analysis confirmed BRCA1 switch off. In each panel, the left panel is representative data of western blot analysis; the right panel shows densitometric analysis. Analysis was performed using three independent experiments. Proteomic approach work flow.
Using 2D gel electrophoresis, we obtained a gel map of about protein spot for each replicate Figure 3. Image analysis allowed to identify 25 differentially expressed proteins.
Of these, 15 protein spots were down-expressed, while 10 were up-regulated. Gel images were analyzed using Decider software. Numbered spots indicate proteins that have statistically significant differential expression between samples according to Image master 2D Platinum 7. A list of identified protein is provided in Table 3. In the table are reported mass spectrometry identifications for protein spots that resulted differentially expressed following 2DE analysis.
For each spot are reported: spot id, gene name, identification score, number of unique peptides, isolectric point pI , and molecular weight MW. In the last columns are reported quantitative data. Western blot analysis confirming 2DE data for gelsolin a and prohibitin b. Images were acquired using the Alliance 2. In each panel, the left panel is representative of western blot analysis; the right panel shows densitometric analysis.
Differentially expressed proteins were connected using ingenuity pathway analysis Ingenuity Systems, www. Data sets with protein identification and quantitative data were loaded into the application that creates hypothetical networks of protein interaction based on ingenuity pathway knowledge base.
IPA software generates a list of networks in function on their connectivity, allowing to associate specific biological functions to genes here included [ 23 , 27 , 29 , 32 ]. Proteins were mapped onto three networks Figure 5. The most representative, with a score of 28 and 13 focus molecules, had function associated with cancer, cellular movement, and connective tissue disorders. The second network, including 12 focus molecules with a score of 25, exhibited functions connected with free radical scavenging, protein synthesis, DNA replication, recombination, and repair.
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