This strategy is a powerful tool for comprehensively studying the global changes of plant phosphoproteomes, in response to biotic or abiotic stresses. This technique allows in-depth phosphoproteomics analysis, in an easy-to-use and high throughput manner. For IMAC stage tip preparation, use a 16 gauge blunt ended needle, to penetrate a propylene frit disc, and use a plunger to gently press the frit into the tip.
Then, place an inverted nickel-NTA spin column, onto a 1.5 milliliter tube, and use the plunger to press the frit of nickel-NTA beads gently into the tube. For phosphopeptide enrichment, using an IMAC stage tip, add 400 microliters of loading buffer to the nickel-NTA beads, and load the entire volume of bead solution into one IMAC stage tip. Pass the solution through the tip by centrifugation and load 100 microliters of 50 millimolar EDTA, to strip the nickel ions from the tip by centrifugation.
Treat the tip with 100 microliters of loading buffer by centrifugation. Next, treat the tip with 100 microliters of 50 millimolar ferric chloride in 6%acetic acid, by centrifugation. Condition the stage with 100 microliters of loading buffer, by centrifugation.
Followed by centrifugation with 100 microliters of loading buffer, supplemented with the sample peptides. After centrifugation, rinse the tip two times, with 100 microliters washing buffer per wash, followed by one rinse with 100 microliters of loading buffer. At the end of of the centrifugation, use scissors to trim the front of the IMAC stage tip and place the trimmed tip inside the high pH, reversed phase tip.
For phosphopeptide fractionation, using a high pH reverse phase C18 stage tip, pass 100 microliters of elution buffer through both layers of stage tips, by centrifugation. At the end of the spin, use tweezers to discard the IMAC stage tip, and add 20 microliters of Buffer A, to the high pH reverse phase stage tip. After centrifugation, add 20 microliters of Buffer 1, to the collected fraction, and collect the eluate in a new tube, by centrifugation.
Repeat the process with Buffers 2 through 8, until all eight fractions have been collected. Then dry the final eluate of each fraction in a vacuum concentrator. In this representative analysis a total of 8, 107 and 7, 248 Phosphopeptides were identified from wild type, and SnRK2 decuple mutant samples respectively, illustrating the efficiency of the workflow in providing in-depth coverage for delineating the global view of signal transduction in Arabidopsis.
Comparison of the number of identified phosphopeptides across eight fractions revealed the presence of few phosphopeptides in the first two fractions, but the majority of phosphopeptides were evenly distributed between the last six fractions. Suggesting this approach provides the ability, to separate complex phosphopeptides from the plant phosphoproteome. Evaluation of the overlap of phosphopeptides between two adjacent fractions, indicated that less than 5%of the phosphopeptides overlap, occurred in the adjacent fractions.
After mannitol treatment, 433 phosphorylation sites were increased in the wild type sample. While, 380 sites were increased in the SnRK2 decuple mutant sample. Among these, 312 phosphosites showed induction in wild type, but not in SnRK2 dec mutant plants.
It is important to use the same amount of pressure to load the frit disc into the tips, to ensure a better reproducibility of the analysis. Soon, cation exchange chromatography can be used, as an alternative measure, for fractionating the enriched phosphopeptides, to obtain a wider coverage of the plant phosphoproteome.
