The Phosphopeptide Shootout: A Study of Reproducibility in IMAC Phosphopeptide Enrichment

摘要

The results of the Phosphopeptide Shootout and the subsequent experiments were disappointing, but are still useable and point to further investigations. The two biggest issues with the results of these experiments are the low enrichment percentages and low overall numbers of phosphopeptides found in each enrichment. Variations in IMAC beads can cause noticeable dierences in enrichment percentages, but overall identications did not seem to depend entirely on the state of the beads. Both old and new beads yield lower results than desired. To improve enrichment percentages, more time in EDTA and FeCl3 incubation times may help. The number of total phosphopeptides detected could also be improved with nano-LC gradients and MS acquisition time. One of the most useful subsequent comparisons would be to systematically vary the dissociation methods used for the MS/MS analysis. HCD and ETD could oer better coverage for phosphopeptides that are present in the enriched sample. Running the samples on high resolution instruments should also provide another interesting comparison to all of this low resolution data. On the processing side of the workow, another comparison would be to search databases using dierent algorithms, such as SEQUEST and MASCOT, to see the performance relative to OMSSA. A third set of experiments, which are described in Figure 4, investigated how the amount of starting material aects overall enrichment. The samples were not fractionated after tryptic digestion as an eort to save time and remove one possible source of variation. These experiments, however, yielded no useful results and are considered a failure of sorts. A future investigation could repeat these experiments, except adding high pH RPLC fractionation to provide a better set of experiments that could provide useful data. The results of this experiment are not conclusive that IMAC enrichment is not viable because one of the researchers that participated in the original shootout later achieved ～5600 phosphopeptide identications and 88% enrichment on 6 mg of fractionated mouse material. This being said, the obvious future comparison is to juxtapose IMAC enrichment with titanium dioxide phosphopeptide enrichment to see if the results can be improved.