This protocol gives you a step-by-step overview on how to prepare beads for affinity cleanup and protein proteolysis and how to use these in determination of low-abundance proteins from dried microsamples. The main advantage is that you're given the tools to prepare and perform highly efficient sampling up of complex samples from very small volumes. This method can be adapted to other protein biomarkers as long as you have access to efficient antibodies to capture either the protein or the proteotypic peptide.
You need to be very careful when you adjust the pH for acid treatment of the antibody, especially if you prepare small volume of beads. To begin, transfer the desired volume of antibody solution to a five-milliliter low-protein-binding microcentrifuge tube, and add a small magnetic stirring bar to the antibody solution. Use a pH meter with a microelectrode to measure the pH, and adjust the pH by first adding 10 microliters of one-molar hydrochloric acid and then reducing the volume as the pH approaches 2.5.
Record the total volume of one-molar hydrochloric acid necessary to adjust the pH to 2.5. Remove the microelectrode, and incubate the acidified antibody on ice, on a magnetic stirrer for one hour. To neutralize the antibody solution, measure the pH and adjust it to seven by first adding 10 microliters of one-molar sodium hydroxide and then reducing the volume of sodium hydroxide as the pH approaches seven.
Record the total volume of one-molar sodium hydroxide added. Mix the magnetic bead suspension thoroughly on a vortex mixer, and withdraw a volume containing the desired amount of bead suspension. For example, to prepare one milliliter of bead suspension, withdraw a volume containing 20 milligrams of beads.
Place the bead suspension on a magnetic rack for one minute, and remove the supernatant. Wash the beads twice with an equal volume of type I water, and mix using a vortex mixer after each addition of the wash solution. Then, place the solution again on a magnetic rack for one minute, and remove the supernatant as shown earlier.
Add acid-treated antibody, 0.5-molar borate buffer, and coupling buffer for antibody coupling to the magnetic beads. Mix using a vortex mixer, and rotate at ambient temperature overnight using an end-over-end sample mixer, preferably with reciprocal rotation and vibration. Then, centrifuge for 10 minutes at 239 g.
Place the tube on the magnet for two minutes, and remove the supernatant. After washing the beads, place the tube again on the magnet for one minute, and follow the same procedure to remove the supernatant. Store this in the desired buffer using the desired stock concentration of beads in a refrigerator.
Allow 10 microliters of serum to be collected in a 10-microliter volumetric absorptive microsampling, or VAMS, tip using the manufacturer's guidelines, and air dry at ambient temperature for at least two hours. Remove the VAMS from the holder, and place it in a two-milliliter low-protein-binding microcentrifuge tube. Add 1, 000 microliters of 100-millimolar ammonium bicarbonate solution, and extract VAMS for one hour at 22 degrees Celsius using a temperature-controlled mixer at 1, 000 rpm.
Transfer the extract to a new 1.5-milliliter low-protein-binding microcentrifuge tube for tryptic proteolysis. Add 30 microliters of washed trypsin beads to each VAMS extract to initiate digestion, and incubate for two hours at 37 degrees Celsius and 1, 000 rpm using a temperature-controlled mixer or similar. Centrifuge at 2, 655 g for five minutes, and transfer the supernatant to a new two-milliliter low-protein-binding microcentrifuge tube.
Add 25 microliters of 14 nanograms per milliliter internal standard, or IS, solution containing the stable isotope-labeled peptide in 100-millimolar ammonium bicarbonate solution. Add two 20 microliters of washed magnetic bead suspension to each low-protein-binding microcentrifuge tube containing a digested VAMS extract and IS.Perform the immunoextraction for one hour using an end-over-end sample mixer at ambient temperature. Wash the magnetic beads by adding 500 microliters of PBS with 0.05%polysorbate 20.
Remove the low-protein-binding microcentrifuge tube containing beads and wash solution from the magnetic rack, and invert carefully until homogeneous. Then, place the low-protein-binding microcentrifuge tube on the magnet for 30 seconds, invert it for 30 seconds, and place it again on the magnet for one minute. Remove the wash solution, and rewash the magnetic beads with PBS, tris hydrochloric acid, and ammonium bicarbonate.
Add 15 microliters of 2%formic acid in type I water to each sample, and incubate for five minutes at 22 degrees Celsius and 1, 000 rpm using a temperature controlled mixer or similar to elute the captured peptides. After placing the eluate on the magnet for one minute, transfer it to a new 1.5-milliliter low-protein-binding microcentrifuge tube. Repeat once, and transfer the second eluate to the same low-protein-binding microcentrifuge tube as the first eluate.
Add 20 microliters of 100-millimolar ammonium bicarbonate to each eluate. Centrifuge it, and transfer 40 microliters of the eluate to microinserts for HPLC vials. In the instrument software, set the column oven temperature to 25 degrees Celsius and the flow rate to 50 microliters per minute.
Then, place the samples in the autosampler. Prepare a sequence containing the samples to be run using the software available for the LC-MS/MS system, and set the injection volume to 10 microliters. Press Run Sequence in the instrument software to start the sequence, and record the peak area of the affinity-captured, ProGRP-specific, tryptic peptide, and its IS.The mass spectrometry, or MS, chromatograms of the proteotypic peptide and the IS after VAMS and dried serum spot, or DSS, sampling, as well as for a spiked serum sample added directly into the extraction solution are shown here.
The representative results of the peak area ratio of the proteotypic ProGRP peptide to IS for serum samples spiked with ProGRP and applied to VAMS, DSS, or directly to extraction solution are shown in this figure. From these results, it can be seen that VAMS provides similar area ratios as the control sample, indicating no loss to the sampling material, while DSS provides a significantly lower area ratio, indicating loss to the sampling material. A comparison of the base peak chromatograms after intact protein extraction and proteotypic epitope peptide extraction is presented in this figure.
When you prepare antibody or enzyme-coated beads yourself, you can tailor the beads to cover your specific needs. This is relevant for targeted protein determination and global proteomics analysis. Sensitive methods in protein determination from VAMS and dried blood spots makes patient-oriented approaches like out-of-hospital testing possible in follow-up of a wider range of diseases.
