2012 Young Investigator Award Finalist: Eugene Ciccimaro

What were the most difficult challenges encountered in this study? And how were they overcome?

The work I highlight describes the use of a full-length isotopically labeled protein used to measure, in a targeted LC–MS assay, endogenous phosphorylation on a group of residues within a key signaling protein. In a first-of-its-kind approach, the labeled full-length protein was expressed using an in vitro expression system and then phosphorylated on tryosin residues via an in vitro kinase reaction. The creation of this standard protein proved extremely difficult and required extensive experimentation in denaturing and refolding of the target protein before the recombinant protein appeared to behave similar to its native form, and reacted with its endogenous kinase target. I ultimately was able to create a suitable standard that could then be used to absolutely quantify endogenous phosphorylation and control for variation in immunoprecipitation.

Which areas of your research did you find the most interesting/enjoyable and why? By comparison, which were the least agreeable?

Learning and devising ways to optimize targeted peptide quantitation assays by LC–MS was (/is) very enjoyable. The speed of data collection and the immediacy with which hypotheses could be drawn and implemented played to my natural abilities as a problem solver, and were a great deal more exciting than my previous experiences in the laboratory, which was much slower paced. Having absolute amounts to work with when describing a biological system governed by phosphorylation was novel, and allowed us to design and conduct unique experiments. Furthermore, the ability to add control to a ‘messy’ experimental process was very rewarding.

In your opinion, how has the research conducted in this study advanced the field of bioanalysis (i.e., future implications)?

Absolute quantification of proteins is becoming a critical aspect of many analytical laboratories. Many critical proteins of interest are at biological concentrations that require some form of preconcentration. To accurately quantify a protein, losses accrued during this and other sample-handling steps should be accounted for. The experimental design highlighted not only shows a way to control for downstream sample preparation, but also allows for absolute quantification to be made on key post-translational modifications (PTMs) on the targeted protein. To my knowledge, this approach is the most analytically rigorous of its kind and, if protein standards were made readily available, could become the future gold standard methodology. The measurement of low level protein phosphorylation on key signaling molecules can serve as a sensitive and specific dosimeter of drug efficacy in cell culture (as we have shown), but may also be used in the future to monitor dose–response in a physiological system. In practice now, it is becoming a very popular technique to use isotopically labeled monoclonal antibody (mAb) to measure mAb targets, the next step in this work is to consider glycosylation and other PTMs on the mAb – this future possibility would build upon my initial work.