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Data to function

A major advantage of the -omics movement is the capacity to sample many features in a single experiment, establishing a global snapshot of the molecules that govern biology. Still, a fundamental challenge remains in establishing the biological relevance for features identified in these large-scale screens. We are developing a high-throughput biochemical assay called multiplexed assay for enzyme specificity (MAES).  MAES will provide an important, and direct link between large-scale -omics assays and biological function.

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MAES description

 

MAES

MAES applies the power of enhanced multiplexing capabilities to in vitro biochemistry. By combining our ultra-plexed peptide tagging strategies with purified components, MAES acts as a matchmaker between enzymes and substrates, thereby turning mere large-scale observations into established protein function.

How MAES works

How MAES works
 

Post-translational modifications (PTMs), such as phosphorylation and acyclations, are ubiquitous throughout the proteome. However, converting large-scale PTM datasets to useful biological information remains a grand challenge. In particular, matching PTMs to the enzymes that regulate their abundance (e.g., kinases and phosphatases) cannot be achieved simply by evaluating whole cell or tissue. To counter this issue, we developed a high-throughput multiplexed assay for enzyme specificity (MAES) that converts discovery PTM data to functional information by en masse substrate-to-enzyme mapping.

MAES can, for example be employed to simultaneously screen multiple kinases and protein substrates. To begin, candidate kinases, ATP, and reaction buffer are added to purified protein(s) in separate but otherwise identical reactions (Figure XX). Following incubation, each sample is digested in preparation for mass spectrometry and labeled with a different isobaric tag. This tag imparts a unique chemical signature to every peptide, linking it to the kinase-substrate reaction from which it originated. Samples are combined and subjected to quantitative tandem mass spectrometry to directly identify peptide sequences and sites of phosphorylation. During peptide fragmentation, the isobaric tags are cleaved, generating reporter ions that are detected in the low-mass region of tandem mass spectra (Figure XX). The intensity of these reporter ions functions as a readout for the abundance of phosphorylated peptides from the corresponding kinase reaction.

Importantly, the MAES approach is applicable to any protein-modifying enzyme (i.e., acetylase, methyltransferase, etc.), making it widely useful in screening for enzymes that modify a substrate of interest. The multiplexing capacity is only limited by the number of isobaric tags in our multiplexing platform. Furthermore, MAES provides a means to rapidly test various experimental parameters (buffer conditions, pH, cofactors, etc.), which can be added or removed from the enzyme reactions to determine their effects.
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Piecing together phosphorylation signaling pathways

  
Piecing together
phosphorylation signaling pathways

MAES is a powerful approach to directly link kinases and phosphatases to their protein substrates. A wide range of purified kinases and phosphatases are already available, and the approach can be adapted to include new kinases and phosphatases provided by collaborators.

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Elucidating epigenetics modifications

Elucidating epigenetics modifications

MAES can be used to connect epigenetic “writers” and “erasers” to established histone marks. These enzymes can include acetyltransferases, deacetylases, methtyltransferases, and demethylases, in addition to the kinases and phosphatases noted above.

MAES Publication Library

MAES Publication Library

 

  

full text
NANOG Is Multiply Phosphorylated and Directly Modified by ERK2 and CDK1 In Vitro

 

 

Contact

For additional information about NCQBCS please send your contact details using the form below.
Note, if you are interested in training, please use the form on our training page.

Contact

For additional information about NCQBCS please send your contact details using the form below.
Note, if you are interested in training, please use the form on our training page.


Where we are

425 Henry Mall, Coon Lab 4462, Madison WI

777 Highland Avenue, Li Lab 5234, Madison, WI

330 N. Orchard Street, Pagliarini Lab 2268, Madison WI

 

How to contact us

Email address: laura.vantoll@wisc.edu