The overall goal of this procedure is to use liquid chromatography, selected reaction monitoring or L-C-S-R-M to quantify the absolute abundance of target proteins within complex biological samples. Following selection of peptide targets, crude external peptide standards are synthesized and used to develop SRM assays using liquid chromatography, mass spectrometry, or LCM ms. The resulting assays are used to perform qualitative lc, SRM analysis of prepared biological samples.
If the biological sample derived target peptide is detected, the biological samples are analyzed using quantitative lc SRM assays by adding a stable isotope dilution series of internal peptide standards. Ultimately, lc SRM can be used to accurately quantify proteins from a wide variety of biological samples and to support investigations of a wide variety of protein targets. The main advantage of this technique over existing methods like immunoassays is that the assay development is relatively quick and inexpensive, and the resulting assays are amenable to multiplexing and not vulnerable to antibody cross reactivity.
To begin prepare lyophilized peptide standards as described in the text protocol. Then add 100 microliters of formic acid acetonitrile solution to each M of Lyophilized peptide standard to produce a peptide concentration of 10 micromolar. Vortex the samples for two minutes and bath.
Sonicate them for five minutes to ensure that peptide dissolution is complete. Pull the dissolved peptides and concentrate the resulting mixture in a vacuum concentrator to a final volume of 80 microliters. Add 20 microliters of acetyl nitrile to dissolve any precipitated peptide.
The sample now contains 10 micromolar of each peptide, as well as 20%volume to volume acetyl nitrile and can be used To prepare the internal and external peptides standards, analyze the mixtures of the external peptide standards at approximately one to 10 picomoles of each peptide per injection by shotgun mass spectrometry using a nano flow HPLC system coupled to a triple quadruple mass spectrometer. As detailed in the text protocol, ensure that each LCMS run includes a 60 minute linear gradient, a column regeneration step, and a column ree equilibration step. Analyze the resulting shotgun data using database, searching against the sequences of the external peptides standards.
Manually review all of the peptide identifications to ensure that they are all unambiguous discarding any ambiguous peptide identifications. Use the shotgun Ms Peptide identifications to construct a spectrum library using a software program such as Skyline Line. Prepare an lc SRM transition list using the three to 10 most intense transitions per precursor ion.
Use the resulting transition lists to perform lc SRM analysis of the mixtures of the external peptides standards. Manually review the resulting L-C-S-R-M data and delete any poorly performing assays. To prepare the biological samples.
First, harvest the cells as described in the text protocol. Add 400 microliters of freshly prepared urea lysis buffer, and mix each sample using gentle pipetting. Transfer each sample to a two milliliter tube that has a screw cap with an O-ring containing approximately 100 microliters of 0.1 millimeters Zirconia silica beads, lyce cells by vortexing the samples for five minutes at full speed bath.
Sonicate the samples for 10 minutes at room temperature to assist homogenization and protein de maturation. Then perform a protein concentration assay of the lysates, such as a bissy acid assay for qualitative analysis. Prepare for 200 microgram aliquots of cell lysate for a stable isotope dilution series.
Add the stable isotope dilution series of the echo molar mixture of the internal peptide standards to the samples. Reduce protein cysteine residues by adding 0.7 microliters of one molar DTT to each sample, and incubating the samples at 60 degrees Celsius for 30 minutes. Alkylate protein cystines by adding seven microliters of buffered ITO acetamide to each sample and incubating the samples at room temperature for 20 minutes in darkness.
For each of the samples, add 482 microliters of 100 millimolar hees adjusted with sodium hydroxide so that the final urea concentration is one molar. Next, triply, digest the proteins into peptides. Add eight microliters of 0.5 micrograms per microliter sequencing grade trypsin to each sample that contains cell lysate.
Then incubate the sample at 37 degrees Celsius for 18 hours. Following incubation, add 440 microliters of 2%volume to volume formic acid centrifuge all of the samples at 21, 000 G for 20 minutes at room temperature to pellet any precipitates that would clog a C 18 SPE cartridge solid phase. Extract each of the SUP natin using a disposable C 18 SPE cartridge burst.
Wet the column by applying one milliliter of buffer B and equilibrate it by applying one milliliter of buffer a twice. Force the mobile phases through the C 18 SPE cartridge using a flat surface to rubber bulb and an extraction manifold. Apply the sample and subsequently wash the cartridge with one milliliter of buffer A twice elute the peptides by applying one milliliter of buffer B.Slowly concentrate each ilu in a vacuum concentrator to a final volume of 100 microliters to evaporate away the acetyl nitrile.
Then add two microliters, the 5%volume to volume formic acid in acetyl nitrile to each sample. Analyze the samples using qualitative L-C-S-R-M as before. For quantitative L-C-S-R-M analysis, run the isotope dilution series of each biological sample shotgun mass spectrometry was used to analyze the external peptide standards.
The top 10 most intense transitions per precursor were selected for L-C-S-R-M. L-C-S-R-M was then used to analyze the external peptide standards. The resulting peptide identifications should be unambiguous.
Qualitative L-C-S-R-M analysis of the biological samples typically resulted in more background signal, but most of the peptide targets were confidently identified. Quantitative L-C-S-R-M was performed using internal peptide standards spiked into the biological samples. The resulting protein abundance values were consistent across the biological replicates and the two target peptides per target protein.
After watching this video, you should have a good understanding of how to develop and perform L-C-S-R-M assays to quantify the absolute abundance of target proteins within complex biological samples.
