The FASP protocol is an interesting approach to your urinary proteomics because of its compatibility with strongly denaturing buffers and because of its ability to concentrate the sample on the filter, which is critical for a diluted protein samples. The FASP protocol coupled with data independent analysis mass spectrometry allows us to achieve deep proteome coverage in EPS-urine, which is urine collected after digital rectal exam. We are currently applying the method you're about to see in a biomarker discovery effort on prostate cancer.
The procedure will be shown by Licia Prestagiacomo, Paola Morelli and Caterina Gabriele. Centrifuge EPS-urine samples within two hours of collection for 10 minutes at 2, 100 RCF at room temperature. Then, store the supernatants at minus 80 degrees centigrade until use.
Throw the samples on ice, or at four degrees. To accelerate the procedure, you can transfer the samples from minus 80 to minus 20 on the day prior to digestion. As stock solutions, you will need 500 millimolar DTT, 10%SDS, one molar Tris buffer at pH eight.
Dilute 500 microliters of each EPS-urine sample with 67 microliters of SDS, 67 microliters of DTT and 33 microliters of Tris buffer. Incubate at 95 degrees centigrade for 10 minutes with gentle shaking. Assemble the centrifugal filter unit with a 10, 000 Dalton molecular weight cut off.
Then, add 300 microliters of diluted EPS-urine sample to the filter. Centrifuge at 14, 000 g for approximately 20 minutes. You may temporarily interrupt the procedure after 15 minutes or so in order to check whether the filtration is proceeding smoothly.
Continue until the whole solution has passed through the filter. Then, add 200 microliters of urea buffer and centrifuge at 14, 000 g for 15 minutes. Repeat this step a second time.
When the flow-through is about to touch the filter, empty the collection Eppendorf by pipetting out the flow-through. Proteins are safe on the filter. For cysteine alkylation, prepare a iodoacetamide solution immediately before use.
Weigh about 10 milligrams of iodoacetamide in a number Eppendorf vial and dissolve it in urea buffer at a concentration of 9.25 milligrams per ml, or in molarity terms, 50 millimolar. Add 50 microliters of iodoacetamide solution to each filter and centrifuge at 6, 000 g for 25 minutes. The lower centrifugation speed will allow the filters not to run dry.
After protein alkylation, wash the filters with two consecutive 200 microliter aliquots of urea buffer and centrifuge at 14, 000 g for 20 minutes. Discard the flow-through. Add 200 microliters of 50 millimolar three ethyl ammonium bicarbonate buffer and centrifuge at 14, 000 g for 20 minutes.
Repeat this step. After fully completing the last bicarbonate wash, transfer the filter unit into a new collection tube. Then, add 60 microliters of 50 millimolar TEAB buffer.
Add one microliter of trypsin solution at a concentration of 200 nanograms per microliter of trypsin. Alternatively, you can prepare the digestion buffer for all samples in an Eppendorf vial and distribute 61 microliters of the digestion buffer to each filter. If you're using a ThermoMixer in order to avoid sample evaporation during overnight incubation, wrap each filter unit with a layer of aluminum foil, and then a layer of parafilm.
Incubate the samples at 37 degrees overnight. On the following morning, transfer the filters to a bench top centrifuge for a very short spin. After spinning, open the Eppendorf lids and add 140 microliters of water.
Centrifuge the vials at 14, 000 g for 25 minutes, in order to collect the peptides. The collected volume should be around 190 microliters. In order to remove traces of SDS from the digest, strong cation exchange purification of the peptides prior to LC-MS-MS is recommended.
Prepare the microcolumn holders by piercing Eppendorf lids with a sharp tool like a pair of tweezers or scissors. The holder will accommodate the microcolumn during centrifugation. Since they are tedious to prepare, holders can be utilized multiple times.
Using a blunt and a needle, remove a plaque of the appropriate extraction disc. Extraction discs are soft polymeric materials containing sorbent particles. In this case, the sorbent is made of particles with strong cation exchange properties.
Accommodate the plaque into a 200 microliter pipette tip. Using a piston push the plug towards the end of the tip. The small disc should be pushed firmly, but avoiding excessive strength.
Insert the tip into the holder and assemble the spin microcolumn using a two milliliter Eppendorf vial from which the original lid was removed. The plug from a gauge 16 needle will have a loading capacity of about five micrograms of peptides. Condition the microcolumn with two consecutive washes.
The appropriate speed will depend on how strongly you push the disc into the pipette tip. Aim at achieving a flowrate in the order of 20 to 30 microliters per minute. Try not to have the tip run dry during washings and during sample loading.
Dilute the sample five fold in wash solution two and load the results in solution at slower speed. Aim for a flowrate of about 15 to 20 microliters per minute. If needed, discard the flow-through.
Wash away residual detergents. Then, allow the disc to run dry before peptide elution. Transfer the microcolumn to a clean 1.5 Eppendorf tube and immediately add the eluent.
Which will be seven microliters of a 500 millimolar ammonium acetate solution containing 20%acetonitrile. Dilute the eluate with 27 microliters of 0.1%formic acid in order to lower the percentage of organic solvent and then inject two microliters of the resulting solution in LC-MS-MS with detection in data dependent mode. Details on the LC-MS setup used in this protocol will be given later in the video.
After performing database search and peptide error integration, calculate the overall peak area. Then, compare it to an external standard curve to estimate peptide concentration in the FASP digest. After estimating peptide amount, purify a new aliquot of FASP digest corresponding to two micrograms of peptides by two consecutive StageTip purifications as described here.
The LC-MS setup used in this protocol is based on direct injection into the analytical column. If you're using a system with a trapping column, you can avoid the step of offline C18 purification. Remember to use dedicated needles and pistons for each chromatographic material.
After eluting the peptides from the C18 Stage Tip with 10 microliters of eluent, partially evaporate the eluate in a vacuum centrifuge for a few minutes, trying to avoid full evaporation. Then, add 47 microliters of 0.1%formic acid and place the resulting solution in an HPLC vial for LC-MS analysis. The system used here is based on direct on column sample loading without using a trapping column.
Analytical columns are in-house made by pulling a 75 micrometer ID capillary after removing a piece of polyamide coating. The resulting tip may be gently shaped with a ceramic cutter. The capillary is then placed into a packing bomb and packed with a 50 milligram per ml isopropanol slurry of C18 three micrometres stationary phase particles.
A gas pressure of between 10 and 20 bars of nitrogen or helium is needed. You may use alternative or commercial chromatography columns running at sub microliter per minute flowrate. Assemble the column on a T-piece.
The other two ends are connected to the high voltage power supply and to the HPLC loop. Assess the optimal spray voltage by running the system at isocratic flow. Then, start your analysis.
Separate the peptides over a two hour long chromatography gradient as displayed. Data acquisition will consist of a quick full MS service scan followed by 26 MS-MS scans on precursor windows of variable width. Starting in 20 m/z width, for the first 20 windows which will cover a m/z range from 350 to 750 Thompson.
Then moving to a 50 m/z width for the following five windows and ending with a single MS-MS scan, having an isolation width of 200 Thompson. Narrower widths account for a more populated region of the spectrum in terms of presence of peptide precursors. For DIA analysis you will need a spectral library.
In order to generate a spectral library, you can perform a few data dependent experiments on the same LC-MS-MS system you have been using for DIA acquisition using the same chromatography gradient. Sample fractionation will increase proteome coverage. Stage Tips can be used for sample fractionation.
Strong cation exchange and basic reverse phase are two valid alternatives. Start with the 10 micrograms of a pool of several FASP digests. Acidify the sample using 0.2%TFA final concentration.
Load the peptides on high capacity C18 Stage Tips. Use multiple discs in case of high peptide amounts. For 10 micrograms of material, two discs are recommended.
Perform C18 purification as described previously. Prepare several vials for eluate collection. As many as the number of fractions.
In this case 10 fractions are produced by stepwise elution. After the last wash, add 20 microliters of the first eluent. 0.2%ammonium hydroxide, 10 millimolar TEAB, 4%acetonitrile.
After fully eluting the first fraction in the first Eppendorf, move the Stage Tip to the next collection vial. Dilute in 20 microliter steps using as eluent, 0.2%of ammonium hydroxide, 10 millimolar TEAB, and then increasing amount of acetonitrile. After fractionating the sample and running data dependent experiments on each fraction, generate your spectral library by database search of MS-MS data.
The library will be then imported in a software capable of DIA data analysis for protein quantification based on a minimum of one unique peptide assigned by a minimum of three transitions. Expect to cover a wide range of the urinary proteome. This figure shows the identification and quantification of proteins across an abundance range, spanning five orders of magnitude.
The workflow here presented combines the concentration advantage and the versatility of the FASP protocol with the sensitivity of DIA scanning mode. This combination provides a rich map of the urinary proteome. Since our analysis setup does not include the use of a trapping column, The FASP digest was purified by both strong cation exchange and reversed phase Stage Tips.
The reversed phase Stage Tips step can be omitted when a trapping column is connected directly to the analytical column. The use of this workflow is recommended for analyzing EPS-urine samples but it's use can be extended to urinary proteomics in general.
