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Fostering new paradigms for the biological sciences

BENJAMIN MINKOFF

Integrated Program in Biochemistry Graduate Program

 

Faculty Advisor: Michael Sussman

bminkoff@wisc.edu


(608) 262-1779

Resume 2012

 

My research lies at the interface of plant signal transduction and analytical chemistry. When Arabidopsis thaliana, the model plant organism, senses it’s losing more water through its leaves than it’s taking up through its roots, it initiates an internal signaling mechanism using abscisic acid (ABA), the major hormone involved in drought response. Ultimately, this results in an altered transcriptome correlated with drought resistance; however, my focus is on the cellular perception pathway that precedes this response. Upon transporting ABA into guard cells (on leaves, which flank stomatal apertures and regulate transpiration-based water loss) as many as 11 of 14 cytosolic receptors (PYR/PYL/RCAR) bind ABA, forming a complex that inhibits at least 6 of 9 clade A protein phosphatase 2Cs (PP2Cs). The inhibition of these phosphatases allows at least 3 of 10 SnRK2 protein kinases to autophosphorylate, activating further kinase activity, which results in ion efflux, stomatal closure, expression of ABA-induced gene families, and ultimately, drought resistance. The complexity, genetic redundancy, and heavy phosphorylation dependence of this pathway make it ideal for proteomic-based approaches, and there are many implications of further detailing plants’ early signaling responses to drought. To study this, I use proteomic-based untargeted mass spectrometry.

Proteomic experiments involve casting a proverbial fishnet into the ocean of complexity that is a cell’s proteome. Highly abundant proteins get “caught” repeatedly during data analyses, and those that exist at low abundance are rarely observed, often yielding data of variable quality. The problem can be said, in part, to be an intrinsic instrumental limitation–a mass spectrometer’s dynamic range is defined as the ratio of highest to lowest abundance/concentration of analytes it can detect. We can neither reproducibly observe nor gather quality data on those that fall outside this range. Thus, it is highly likely there are protein level changes we have yet to observe, for many systems and organisms. Furthermore, this problem is amplified when attempting to perform untargeted phosphorylation analyses–these events can be transient and occur in such low abundance that even observing them can present issues.

One answer to this issue is fractionation prior to introducing a sample onto the mass spectrometer. By far, the most commonly used techniques are based in liquid chromatography (LC). I have already implemented a system of strong cation exchange-based exchange prior to spectral analysis that splits a highly complex sample in a number of less complex fractions, making observation of low abundance peptides and phosphorylation events easier and more reproducible. There are a number of other techniques, LC-based and otherwise, that can augment our experimental pipeline, ranging from density gradient-based cellular centrifugation to complementary fragmentation techniques within the mas spectrometer. These techniques will be used in conjunction with ABA treatment in both wild type and various Arabidopsis mutants to explore the plants’ ABA-altered proteome and phosphoproteome more extensively than has been done before.

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