Encoding substrates with mass tags to resolve stereospecific reactions using Nimzyme.
BACKGROUND
The nanostructure-initiator mass spectrometry based enzyme assay (Nimzyme) provides a rapid method for screening glycan modifying reactions. However, this approach cannot resolve stereospecific reactions which are common in glycobiology and are typically assayed using lower-throughput methods (gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis) often in conjunction with stable isotopically labeled reactants. However, in many applications, library size necessitates the development of higher-throughput screening approaches of stereospecific reactions from crude sample preparations. Therefore, here we test the approach of utilizing Nimzyme linkers with unique masses to encode substrate identity such that this assay can resolve stereospecific reactions.


METHODS
We utilize the nanostructure-initiator mass spectrometry (NIMS) enzyme assay in conjuction with an accurate mass tagging approach where each reactant is tagged with a unique perfluoronated tail. Mass spectrometric analysis was conducted using conventional MALDI-TOF instrumentation.


RESULTS
Stereospecific reaction pathways of three stereoisomers (maltose, lactose and cellobiose) to afford the same product glucose were resolved simutaneously due to the presence of unique fluorous tags on both reactants and products. Not only purified enzymes, but also crude cell lysates can be used in this assay.


CONCLUSIONS
The Nimzyme assay with accurate mass tagging provides a rapid method for screening for targeted stereospecific reactions using mass spectrometry and may be useful for high-throughput screening and functional annotation of a wide range of glycan-modifying enzymes.
Joint BioEnergy Institute, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Emeryville, CA 94608, USA.
Kai
K
Deng
Kevin W
KW
George
Wolfgang
W
Reindl
Jay D
JD
Keasling
Paul D
PD
Adams
Taek Soon
TS
Lee
Anup K
AK
Singh
Trent R
TR
Northen
D002475:Cellobiose / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D005947:Glucose / Q000378:metabolism; D007785:Lactose / Q000378:metabolism*; D008320:Maltose / Q000378:metabolism*; D049329:Nanostructures / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013237:Stereoisomerism; D013379:Substrate Specificity
D002475:Cellobiose; D008320:Maltose; D005947:Glucose; D007785:Lactose