The overall goal of the following procedure is to isolate and quantify peptides in a complex mixture that act as surrogates for their respective protein. First large proteins within the mixture are digested to component peptides using an enzyme such as trypsin. Then the targeted peptide analytes and a spiked stable isotope labeled internal standard are enriched using antip peptide antibodies, immobilized on protein G coded magnetic particles following isolation.
The targeted peptides are alluded from the magnetic particles and analyzed by mass spectrometry for quantitation relative to the internal standard. The main advantages of this technique over existing methods like traditional immunoassays is that it is faster and more cost effective in building large numbers of assays. It has a high success rate and it is readily multiplexed demonstrating the procedure will be laid out.
A technician in the laboratory Haw a 10 microliter plasma Eloqua on wet ice. Transfer the sample to a 1000 microliter deep well plate and cover with parable film to denature the proteins at 20 microliters of fresh nine molar urea 30 millimolar DTT to each sample and incubate for 30 minutes At 37 degrees Celsius, add three microliters of fresh 500 millimolar I oto acetamide to each sample and incubate for 30 minutes in the dark at room temperature. Now dilute the urea to 0.6 molar with 100 millimolar tris pH eight, and add 10 microliters of one microgram per microliter trips and stock solution for a one to 50 enzyme to substrate ratio.
After incubating the reaction at 37 degrees Celsius overnight at three microliters of neat formic acid to a final concentration of 1%continue to add the stable isotope standard or multiple standards. If performing a multiplex assay wash the Oasis cartridge plate well with 500 microliters of 0.1%formic acid in 80%ACE nitrile discarding the flow through. Then equilibrate the cartridge plate by adding 500 microliters of 0.1%formic acid in water and discard the flow through.
Load the digest samples onto the cartridge plate and adjust the vacuum so the flow is very slow. Wash three times with 500 microliters of 0.1%formic acid in water and discard the flow through elute the peptides twice with 500 microliters of 0.1%formic acid in 80%aceto trial, then lyophilize or speed back the eluate to dryness and reconstitute the dried peptides in 50 microliters of PBS plus 0.03%Chaps transfer the samples to a standard Kingfisher 96 well plate add one microgram of antibody and 1.5 microliters of protein G coded magnetic beads per target. Vortex the beads prior to addition.
To ensure that the beads are well suspended. Cover the plate with foil and incubate overnight with gentle tumbling centrifuge. The plate at 400 RPM for 10 seconds to remove any liquid from the foil surface.
Remove the foil cover. Sample manipulation occurs on an automated kingfisher platform on the Kingfisher platform. Wash the beads twice with 200 microliters of PBS plus 0.03%chaps and once with 200 microliters of a one to 10 dilution of PBS plus 0.03%Chaps now elude the peptides with 25 microliters of 5%acetic acid plus 0.03%chaps.
Place the elution plate on a magnet and transfer the eluate to a 96 well plate taking care not to transfer the leftover beads. Cover the plate with the ceiling mat, deliver this plate containing the eluate to the triple, quadruple mass spectrometer for analysis. The MRM method is constructed by obtaining a tandem mass spectrum of the peptide of interest than choosing and optimizing the best transition fragment ions.
Typically, a configuration for MRM analysis uses a C 18 trap and analytical columns with two mobile phases. Load 10 microliters of the sample and elute by a linear gradient. Integrate peak areas for the light and heavy peptides, and calculate the peak area ratio of unlabeled to labeled peptide in each sample.
The light and heavy peptides elute at the same time and multiple transitions can be monitored for each peptide To confirm the identity, the peak area of the endogenous peptide is measured relative to the area of the internal standard to provide a quantitative measure of the target peptide. After watching this video, you should have a good understanding of how to implement this technique in your laboratory. It can be used to quantify any protein of interest, making it suitable for a broad range of applications in biological research.
