In the realm of proteomics, it is important to minimize the time needed for biochemical processing as well as data acquisition by mass spectrometry. Different steps in a process are difficult to monitor and if an experiment fails, one may not know until it has already taken up a significant amount of time on the bench and in the instruments. This poster will display how protein and peptide fluorophore-quencher moieties can prove to be useful tools in monitoring processing steps including protein digestion, solid phase extraction, and IMAC enrichment. An EnzChek peptide and protein-based substrate were each serially diluted, introduced to trypsin, and run through a SpectraMAX spectrophotometer. Samples were then passed through C18 stage tips and the flow through, washes, and eluate were read for fluorescence on the plate reader. Upon digestion with trypsin, the peptide and protein showed an increase in fluorescence signal intensity over time. Signal plateau indicated that the digestion had reached completion. Peptide samples were cleaned with C18 stage tips as well as Agilent RP-S tips. The retention of the fluorescent peptide can be used as a proxy for sample retention. Mass spectrometry will be performed to disprove any concerns of the substrate signal dampening the signal of the samples. Dyes such as rhodamine are being considered to insert as an internal standard to account for any signal fluctuation. The peptide and protein moieties proved to be useful tools to apply to assays in order to increase monitoring capabilities and efficiency. Multiple moieties with different excitation/emission wavelengths will be implemented to monitor multiple steps in the protocol for the P100 assay under the LINCS project.

Fluorescent tracers used to track the efficiency of an automated phosphopeptide workflow

251st National Meeting (2016)

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