Here, we describe an instructional method based on a fast heat inactivation of protease to preserve natural intracellular peptides, avoid degradation in cells and tissues, followed by relative quantitation of peptides using isotopic labeling. This labeling method has so much advantages. The reagents are commercially available, inexpensive, chemically stable, and allows the analysis of up to five samples in a single serum.
Begin by cultivating human neuroblastoma cells in a 15 centimeter dish in DMEM containing 15%FBS and 1%penicillin streptomycin. Maintain the cells at 37 degrees Celsius under 5%carbon dioxide. Wash the cells twice with PBS, then add 10 milliliters of PBS, scrape the cells, and collect them into a 15 milliliter tube.
Centrifugate the cells at 800 times G for five minutes and remove the supernatant. Resuspend the pellet in one milliliter of ultra purified deionized water at 80 degrees Celsius, then transfer the contents of the tube to a two milliliter microfuge tube. After heat inactivation of zebrafish, collect the whole brain in a two milliliter microfuge tube and freeze it at minus 80 degrees Celsius until analysis.
Resuspend the tissue sample in one milliliter of ultra purified deionized water at 80 degrees Celsius and sonicate with a probe using 30 pulses of one second. Incubate the cellular lysate or homogenate tissue at 80 degrees Celsius for 20 minutes, then cool it on ice for 10 to 30 minutes. Add 10 microliters of one molar hydrochloric acid stock solution for each one milliliter of sample volume.
Mix by vortexing for 20 seconds and incubate on ice for 15 minutes. Centrifugate the cellular lysate or the homogenate tissue at 12, 000 times G in four degrees Celsius for 15 minutes. Collect the supernatant in low binding protein microcentrifuge tubes and store it at minus 80 degrees Celsius.
Clean the ultrafiltration devices with water and centrifuge at 2, 300 times G for three minutes, then discard the water from the ultrafiltration device. Pipette the supernatant into a pre-washed 10 kilodalton cutoff filter and spin in a refrigerated centrifuge at four degrees Celsius. Check the pH of the sample.
Equilibrate the column with one milliliter of 100%acetonitrile, then wash it with one milliliter of 5%acetonitrile with 0.1%trifluoroacetic acid. Load the complete volume of the sample in the column and wash the column with one milliliter of 5%acetonitrile with 0.1%trifluoroacetic acid. Elute the peptides from the column with 1.8 milliliters of 1%acetonitrile with 0.15%trifluoroacetic acid into protein low binding microcentrifuge tubes.
Dry the sample entirely in a vacuum centrifuge at 30 degrees Celsius and monitor the concentration time on display. Store the sample at minus 80 degrees Celsius. Resuspend the peptide samples in 100 to 200 microliters of ultra purified water and pipette 2.5 microliters of the standard peptide concentrations and samples onto the white 96-well plate.
Add 25 microliters of 0.2 molar phosphate buffer and 12.5 microliters of fluorescamine. Homogenize gently for one minute on the orbital rotator. Next, add 110 microliters of water to stop the reaction.
Adjust the settings on the spectrofluorometer, then read the plate. Add 1/10th volume of one molar trimethylammonium bromide to each peptide sample with up to 25 micrograms of the peptide. Ensure that the pH is between five and eight, adjusting with hydrochloric acid or sodium hydroxide, if needed.
Add four microliters of non-deuterated, deuterated formaldehyde or carbon-13 deuterated formaldehyde. Mix for five seconds by vortexing. Add four microliters of sodium cyanoborohydride or deuterated sodium cyanoborohydride to the peptide sample.
Then mix the sample for five seconds by vortexing. Incubate the peptide sample in a fume hood for two hours at room temperature, vortexing every 30 minutes. Repeat the addition of non-deuterated, deuterated, or carbon-13 deuterated formaldehyde and sodium cyanoborohydride or deuterated sodium cyanoborohydride, vortexing after each addition.
Incubate the samples in the fume hood overnight at room temperature. Add 16 microliters of ammonium bicarbonate and mix by vortexing. Place the sample on ice.
Add eight microliters of 5%formic acid and vortex for another five seconds. Combine the peptide samples, adjust the pH between two and four, then desalt the combined samples on reversed phase cleanup columns using acetonitrile and trifluoroacetic acid as previously described. Dry the sample entirely in a vacuum centrifuge at 30 degrees Celsius, then store it at minus 20 degrees Celsius.
The labeled peptides are observed using mass spectra. Red arrows indicate the presence of peak pairs of different peptides labeled with two isotopic forms, L1 and L5, for comparison between two different samples S1 and S2 respectively. The mass spectrum of a peptide present in three different samples, S1, S2, and S3, labeled with L1, L3, and L5 tags respectively is shown here.
A quadruplex labeling was performed. Control samples S1 and S3 were labeled with L1 and L3 respectively and compared with two experimental samples, S2 and S4 labeled with L2 and L4.No significant difference was observed. The isotopic labeling was used to show substrates in products in vitro for a given protease or peptidase.
A peptide that does not change in the presence of the neurolysin enzyme is shown here. Peptides that disappear or reduce in the presence of the enzyme are substrates, while those that increase are considered products. The figure is an example of identifying a peptide sequence performed by a database search engine labeled with three different forms of tags.
Before it's defined, ensure the sample is completely cool to avoid breaking the peptide bonds caused by hard acidification. In addition, the cleaning of ultrafiltration devices is essential. mass spectrometry is an excellent method to quantify peptides between different experimental conditions and for analysis studies by identifying peptidase substrates.
