Nitropeptide Profiling and Identification Illustrated by Angiotensin II

This message that can help answer key questions about how to enrich and identify nitropeptides by a chemical approach and the mass spectrometry. This technique has an enhanced sensitivity for detecting nitropeptides by mass spectrometry and that can be applied to both in vitro biochemical systems and endogenous biological tissues. For nitro-angiotensin II desalting, use a one milliliter pipet to precondition a reverse-phase solid-phase extraction or SPE column with the addition of 500 microliters of methanol and 500 microliters of water into the top of the column.

When all of the water has run through the column, load 410 microliters of nitro-angiotensin II solution onto the column. Wash the column with 500 microliters of 5%methanol and water and 300 microliters of 80%methanol and water to elute the nitropeptide. Then dry the eluate by speed vacuum in the default setting at room temperature.

For primary amine alkylation, reconstitute the powdered nitro-angiotensin II in 100 microliters of 100 millimolar Triethylammonium bicarbonate solution and add 400 microliters of 4%formaldehyde to the solution in a fume hood with brief mixing. Next, add four microliters of 0.6 molar sodium cyanoborohydride to the solution with shaking at 400 rotations per minute for one hour at room temperature. At the end of the incubation, quench the labeling reaction with 16 microliters of 1%ammonia solution for five minutes at room temperature followed by acidification with eight microliters of formic acid.

Then desalt the solution in a new reverse-phase SPE column as just demonstrated. For nitrotyrosine to aminotyrosine reduction, reconstitute the dimethyl-labeled nitro-angiotensin II in 500 microliters of PBS and add 10 microliters of one molar sodium dithionate for a one hour incubation at room temperature. After the reduction reaction, the yellow solution will become clear.

Desalt the reduced sample in a new reverse-phase SPE column as demonstrated. For biotinylation and enrichment, reconstitute the dimethyl-labeled aminoangiotensin II in 500 microliters of PBS followed by the addition of five microliters of 40 nanomolar NHS-S-S biotin dissolved in dimethyl sulfoxide for a two hour incubation at room temperature. At the end of the incubation, quench the reaction with one microliter of 5%hydroxylamine and add 500 microliters of fresh PBS to 100 microliters of Streptavidin Agarose beads for the equilibration by three separate centrifugations.

After the last centrifugation, add 100 microliters of beads to the reaction system for a one hour incubation on a Rotary Shaker at room temperature. At the end of the incubation, wash the system four times with 500 microliters of fresh PBS per wash followed by the addition of 400 microliters of 10 millimolar dithiothreitol for a 45 minute incubation at 50 degrees Celsius. At the end of the incubation, spin down the column and transfer the supernatant into a new tube containing 20 microliters of 0.5 molar iodoacetamide for a 20 minute incubation in the dark.

Then desalt the solution in a new reverse-phase SPE column as demonstrated and resolve the dry peptide in 20 microliters of 0.1%formic acid. For detection, load the product onto a liquid chromatographer with a tandem mass spectrometer. Separate the modified angiotensin II peptides by a 60 minute gradient elusion at a flow rate of 0.3 microliters per minute with the nano-high pressure liquid chromatography system that is directly interfaced with the high-resolution mass spectrometer.

In the data-dependent acquisition mode, set a single full scan mass spectrum in the mass spectrometer, followed by 20 data-dependent tandem mass spectrometer scans at 28%normalized collision energy. Then open the mass spectra data and identify the peaks of the product in each step to confirm the chemical reaction has been successfully performed. Here, representative mass spectra of angiotensin II before and after each chemical modification as demonstrated can be observed.

The molecular weight of the compound is indicated by the mass-to-charge ratio values of the mono isotope peak, indicating that the chemical modification on angiotensin II was successfully achieved for that step. Here, the final product as detected and characterized by liquid chromatography with tandem mass spectrometry is shown. Quantitative analysis of the nitropeptides by dimethyl-labeling allows calculation of the relative amounts of light and heavy via comparison of the intensity of the mono isotope peak in each group, allowing quantification of the enriched nitropeptides from different groups.

Following the procedure, you can use a program search engine such as the SEQUEST max count of pFIND to identify the potential nitropeptides. After its development, this technique paved the way for the starting of the biological functions of specific nitration sites.