The overall goal of the following experiment is to achieve the largest number of protein identifications from the cochlear sensory epithelium using various proteomic techniques. This is achieved by isolating cochlea, sensory epithelial from adult mice and sonicate in lysis buffer to obtain protein as a second step. Different digestion strategies are applied to whole lysate or to fractionated protein lysate in order to enhance peptide and protein sequence coverage.
Next different separation techniques are utilized, including strong cation exchange and reversed phase chromatography to reduce sample complexity prior to mass spec analysis. The results show identification of a large number of membrane and soluble proteins for mouse cochlea sensory epithelium by using multiple proteomics approaches and a high resolution mass spectrometer. The main advantage of this technique over existing methods like antibody microarray and two dimensional gel electrophoresis is that a large number of both soluble and membrane proteins are identified, hence increasing the cochlear proteome data.
Though this method can provide insight into biomarkers related to hearing and tearing impairments, it can also be applied to other systems such as identification of biomarkers related to age and breast or lung cancer. This method can also be applied to studying other biological samples such as blood. We first had the idea for this method when we observed the limited proteomic dataset available from the mammalian inner ear, and in particular, the limited number of membrane proteins that have been identified Prior to starting this procedure.
Isolate cochlear sensory epithelium from postnatal day 30 mice. After washing the tissue with one x phosphate buffer, add 100 microliters of lysis buffer to the tissue and sonicate the mixture for 30 seconds on ice using a sonic dismember. Cool the lysate on ice for one minute between each sonication, then sonicate the sample a total of three times.
Centra fused the suspension at 16, 000 times G at four degrees Celsius for five minutes. When finished, retain the supernatant for digestion. Following this, add a 30 microliter aliquot of cochlear protein extract directly to a 30 K spin filter and mix with 200 microliters of eight molar urea in Riss HCL centrifuge.
The mixture at 14, 000 times G for 15 minutes. When finished, dilute the concentrate with 200 microliters of eight molar urea solution and repeat centrifugation using the same conditions as before. Next, add 10 microliters of 10 XI oto acetamide, and 90 microliters of eight molar urea solution to the concentrate in the filter and vortex for one minute.
After placing the spin filter in a micro centrifuge rack, incubate for 20 minutes at room temperature in the dark following centrifugation at 14, 000 times G for 10 minutes at 0.1 micrograms per microliter of endo protease ly C in a one to 50 enzyme to protein ratio, and incubate the mixture overnight at 30 degrees Celsius. When finished, add 40 microliters of 100 millimolar ammonium bicarbonate solution to the filter unit and centrifuge the mixture at 14, 000 times G for 10 minutes After repeating the previous step, wash the filter unit with 40 microliters of eight molar urea. Then wash the filter unit two times with 40 microliters of milli Q water.
Once the filter unit has been washed three times with 100 microliters of 50 millimolar ammonium bicarbonate solution add 0.4 micrograms per microliter of trypsin in a one to 100 enzyme to protein ratio, and incubate the mixture overnight at 37 degrees Celsius. Following incubation, elute the triptic peptides by adding 40 microliters of 50 millimolar ammonium bicarbonate solution to the filter unit and centrifuge the mixture at 14, 000 times G for 10 minutes. Then repeat this step one more time.
At this point, inject 50 to 100 micrograms of digested protein sample onto a 200 by 2.1 millimeter five micrometer strong cation exchange column To separate the peptides, monitor the peptide fractions at 280 nanometers and collect the fractions in two minute intervals. Using a fraction collector, then dry the fractions in a vacuum concentrator. When finished, store the dried fractions at minus 80 degrees Celsius until ready to use for liquid chromatography.
Tandem mass spectrometry analysis. In another approach, gel free fractionation was used to separate the protein mixture. Once the cochlear protein supernatant has been desalted by acetone precipitation, add 30 microliters of five x sample buffer to the desalted protein previously suspended in 112 microliters of Milli Q water.
Next, add eight microliters of one molar DTT and heat the mixture at 95 degrees Celsius for five minutes to reduce the protein disulfide bonds. After heating allow the mixture to cool. Add eight milliliters of running buffer to the anode reservoir, 150 microliters of running buffer to the sample collection chamber, and six milliliters of running buffer to the cathode reservoir.
Following this load, 150 microliters of the cochlear protein mixture into the sample loading chamber, using an 8%tris acetate cartridge with a mass range of 3.5 to 150 kilodaltons. After starting the run, collect the sample from the sample collection chamber at each paused interval on the instrument using a pipette and add to a freshly labeled micro tube. Then wash the collection chamber two times with 150 microliters of running buffer and discard.
Following 1D gel electrophoresis of the gel free fractions. Add each individual fraction directly to a 30 K filter unit. Then mix each fraction with 200 microliters of eight molar urea in tris HCL following centrifugation at 14, 000 times G for 25 minutes, add 10 microliters of 10 XI oto acetamide in urea solution to the concentrate in each filter unit and vortex for one minute.
After placing the spin filters in a micro centrifuge rack, incubate the samples for 30 minutes at room temperature in the dark. Next, add 0.1 micrograms per microliter of LY C for a one to 50 enzyme to protein ratio to the filter units following overnight incubation at 30 degrees Celsius, add 40 microliters of 100 millimolar ammonium bicarbonate solution to the spin filters and centrifuge the samples at 14, 000 times G for 10 minutes after repeating the previous step one time, wash the spin filters three times with 100 microliters of 50 millimolar ammonium bicarbonate solution add 0.4 micrograms per microliter of trypsin in a one to 100 enzyme to protein ratio after the final wash. Once the samples have been incubated overnight at 37 degrees Celsius elute the triptych peptides from each filter unit by adding 40 microliters of 50 millimolar ammonium bicarbonate solution and centrifuge the samples at 14, 000 times G for 10 minutes.
After repeating this, step one time, analyze the samples by liquid chromatography tandem mass spectrometry. There were two double digestion procedures used. The first was a triptych digestion, followed by a second digestion with trypsin, which were pooled fractionated on a strong cation exchange column into 18 fractions, and analyzed by nano liquid chromatography tandem mass spectrometry.
A total of 1, 485 proteins were identified with a 1%FDR when performing a single run liquid chromatography tandem mass spectrometry. Using this experimental approach. Among the identified proteins 324 and 258 proteins were categorized in mitochondrion and plasma membrane respectively.
The second double digestion procedure consisted of lys C digestion, followed by trypsin digestion. Each digest was individually loaded and separated on the strong cation exchange column into 18 fractions, and analyzed by nano liquid chromatography tandem mass spectrometry. The results of the LY C and trypsin digestions produced a total of 3, 503 proteins with a 1%FDR.
Additionally, 605 and 617 proteins were categorized in mitochondrion and plasma membrane respectively. This approach provided the largest number of membrane associated protein IDs. Duplicate analysis of the LY C and trypsin.
Fractions showed more than 65 of the proteins identified were shared between the experiments. However, there were also newly identified proteins in the replicate analysis. The additional peptides and proteins were identified due to small changes in chromatography, therefore leading to different peptides for fragmentation.
A silver stain gel was prepared to visualize the results from gel free fractionation as illustrated. Here, the gel showed protein separation by increasing molecular weight for each consecutive fraction. Therefore, the 12 gel free liquid fractions were digested using two different multifacet digestion approaches and analyzed by liquid chromatography tandem mass spectrometry.
The first digestion approach was performed using a double trypsin digestion, which led to the identification of 2, 165 proteins with a 1%FDR when performing a single run liquid chromatography tandem mass spectrometry. In this experiment, 516 and 399 proteins were categorized in mitochondrion and plasma membrane respectively. The second digestion approach was performed using endo proteinase ly C followed by trypsin digestion, single run liquid chromatography.
Tandem mass spectrometry analysis identified 2, 211 proteins with a 1%FDR. This approach showed a similar number of membrane associated proteins as when using the trypsin trypsin approach, the mass spectrometry proteomics data have been deposited to the Proteome Exchange Consortium with the dataset identifier PXD 0 0 0 2 3 1. While attempting this procedure, it is important to remember to avoid contamination at every step and keep the tissue on ice when performing protein extraction to avoid protein degradation.
